Pcsk9 antagonists

ABSTRACT

The present invention provides antagonizing antibodies, antigen-binding portions thereof, and aptamers that bind to proprotein convertase subtilisin kexin type 9 (PCSK9). Also provided are antibodies directed to peptides, in which the antibodies bind to PCSK9. The invention further provides a method of obtaining such antibodies and antibody-encoding nucleic acid. The invention further relates to therapeutic methods for use of these antibodies and antigen-binding portions thereof to reduce LDL-cholesterol levels and/or for the treatment and/or prevention of cardiovascular disease, including treatment of hypercholesterolemia.

This application is a divisional of U.S. application Ser. No.13/225,265, filed Sep. 2, 2011, which is a divisional of U.S.application Ser. No. 12/558,312, filed Sep. 11, 2009, now issued as U.S.Pat. No. 8,080,243, which claims priority, under 35 USC §119(e), to thefollowing US provisional applications, U.S. Appl. No. 61/096,716, filedSep. 12, 2008, U.S. Appl. No. 61/232,161, filed Aug. 7, 2009, and U.S.Appl. No. 61/235,643, filed Aug. 20, 2009.

REFERENCE TO SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted sequence listing in .txt format. The .txtfile contains a sequence listing entitled “PC33718D_SequenceListing.txt”created on Feb. 11, 2013 and having a size of 52 KB. The sequencelisting contained in this .txt file is part of the specification and isherein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies, e.g., full lengthantibodies or antigen-binding portions thereof, peptides, and aptamersthat antagonize the activity of extracellular proprotein convertasesubtilisin kexin type 9 (PCSK9), including its interaction with the lowdensity lipoprotein (LDL) receptor (LDLR). More specifically, theinvention relates to compositions comprising antagonist PCSK9antibodies, peptides, and/or aptamers and methods of using theseantibodies and/or peptides and/or aptamers as a medicament. Theantagonist PCSK9 antibodies, peptides, and aptamers can be usedtherapeutically to lower LDL-cholesterol levels in blood, and can beused in the prevention and/or treatment of cholesterol and lipoproteinmetabolism disorders, including familial hypercholesterolemia,atherogenic dyslipidemia, atherosclerosis, and, more generally,cardiovascular disease (CVD).

BACKGROUND OF THE INVENTION

Millions of people in the U.S. are at risk for heart disease andresulting cardiac events. CVD and underlying atherosclerosis is theleading cause of death among all demographic groups, despite theavailability of therapies directed at its multiple risk factors.Atherosclerosis is a disease of the arteries and is responsible forcoronary heart disease associated with many deaths in industrializedcountries. Several risk factors for coronary heart disease have now beenidentified: dyslipidemias, hypertension, diabetes, smoking, poor diet,inactivity and stress. The most clinically relevant and commondyslipidemias are characterized by an increase in beta-lipoproteins(very low density lipoprotein (VLDL) and LDL) with hypercholesterolemiain the absence or presence of hypertriglyceridemia (Fredrickson et al.,1967, N Engl J Med. 276:34-42, 94-103, 148-156, 215-225, and 273-281).There is a long-felt significant unmet need with respect to CVD with60-70% of cardiovascular events, heart attacks and strokes occurringdespite the treatment with statins (the current standard of care inatherosclerosis). Moreover, new guidelines suggest that even lower LDLlevels should be achieved in order to protect high risk patients frompremature CVD [National Cholesterol Education Program (NCEP), 2004].

PCSK9, also known as NARC-1, was identified as a protein with a geneticmutation in some forms of familial hypercholesterolemia. PCSK9 issynthesized as a zymogen that undergoes autocatalytic processing at themotif LVFAQ in the endoplasmic reticulum. Population studies have shownthat some PCSK9 mutations are “gain-of-function” and are found inindividuals with autosomal dominant hypercholesterolemia, while other“loss-of-function” (LOF) mutations are linked with reduced plasmacholesterol. Morbidity and mortality studies in this group clearlydemonstrated that reducing PCSK9 function significantly diminished therisk of cardiovascular disease.

Of significant importance to the treatment of CVD, a LOF mutation maysensitize humans to statins, allowing for efficacy at a lower dose(hence, improving risks associated with safety and tolerance) andpotentially achieving lower plasma cholesterol levels than with currenttherapies.

PCSK9 is secreted into the plasma predominantly by hepatocytes. Geneticmodulation of PCSK9 in mice confirmed the ability of PCSK9 to regulateblood lipids, and suggested that it acts to down-regulate hepatic LDLRprotein levels.

The mechanism by which, and the site at which, PCSK9 down-regulates LDLRprotein has not been clearly established. When over-expressed, PCSK9 mayact both within the hepatocyte and as a secreted ligand for LDLR. Thereis strong evidence that extracellular PCSK9 binds to cell surface LDLRand promotes LDLR degradation at an intracellular site. However, it isalso possible that PCSK9 could interact with the LDLR when the twoproteins are translated within the endoplasmic reticulum (ER) andtraffic through endosomal compartments towards the cell membrane.Maxwell et al., 2005, Curr. Opin. Lipidol. 16:167-172, showed thatPCSK9-mediated LDLR endocytosis and degradation was not altered byproteosome inhibitors nor was it modulated by different classes oflysosomal and nonlysosomal proteases. Two naturally occurring familialhypercholesterolemia mutations, S127R and D129G, have been reported tobe defective in autoprocessing and secretion as levels of these mutantproteins were greatly reduced or undetectable in the media oftransfected cells. Yet these mutants demonstrated an enhanced ability todown-regulate LDLR, consistent with their identification in individualswith high plasma LDL (Homer et al., 2008, Atherosclerosis 196:659-666;Cameron et al., 2006 Human Molecular Genetics 15:1551-1558; Lambert etal., 2006, TRENDS in Endocrinology and Metabolism 17:79-81. Since thesemutants apparently do not get secreted extracellularly, and yet dodownregulate LDLR, this strongly suggests that an intracellular site ofaction is physiologically important.

From the information available in the art, and prior to the presentinvention, it remained unclear whether the introduction of an antibody-,peptide-, or aptamer-based PCSK9 antagonist into the blood circulationto selectively antagonize extracellular PCSK9 would be effective toreduce hypercholesterolemia and the associated incidence of CVD and, ifso, what properties of a PCSK9 antagonist are needed for such in vivoeffectiveness.

SUMMARY OF THE INVENTION

This invention relates to antagonist antibodies, peptides, and aptamersthat selectively interact with and inhibit PCSK9 function. It isdemonstrated for the first time that certain PCSK9 antagonists areeffective in vivo to lower blood cholesterol.

In one embodiment, the invention provides an isolated antagonist ofPCSK9 which comprises an antibody, a peptide, or an aptamer, whichinteracts with PCSK9 and when administered to a subject lowers theLDL-cholesterol level in blood of said subject. The antagonist can be anantibody, for example, a monoclonal antibody or human, humanized, orchimeric antibody.

In another embodiment, the invention provides an isolated anti-PCSK9antibody which specifically binds to PCSK9 and which is a fullantagonist of the PCSK9-mediated effect on LDLR levels when measured invitro using the LDLR down regulation assay in Huh7 cells disclosedherein.

In yet another embodiment, the invention provides an isolated antibodywhich antagonizes the extracellular interaction of PCSK9 with the LDLR,as measured by PCSK9 binding to the LDLR in vitro, and, whenadministered to a subject, lowers the LDL-cholesterol level in blood ofsaid subject. Preferably, the antibody recognizes an epitope on humanPCSK9 that overlaps with more than about 75% of the surface on PCSK9that interacts with the EGF-like domain of the LDLR as described in Kwonet al., 2008, PNAS, 105:1820-1825.

In yet another embodiment, the invention provides an antibody thatrecognizes a first epitope of PCSK9 that overlaps with a second epitopethat is recognized by a monoclonal antibody selected from the groupconsisting of 5A10, which is produced by a hybridoma cell line depositedwith the American Type Culture Collection and assigned accession numberPTA-8986; 4A5, which is produced by a hybridoma cell line deposited withthe American Type Culture Collection and assigned accession numberPTA-8985; 6F6, which is produced by a hybridoma cell line deposited withthe American Type Culture Collection and assigned accession numberPTA-8984, and 7D4, which is produced by a hybridoma cell line depositedwith the American Type Culture Collection and assigned accession numberPTA-8983.

In another embodiment, the invention provides an antibody to humanPCSK9, wherein the antibody recognizes an epitope on human PCSK9comprising amino acid residues 153-155, 194, 195, 197, 237-239, 367,369, 374-379 and 381 of the PCSK9 amino acid sequence of SEQ ID NO: 53.Preferably, the antibody epitope on human PCSK9 does not comprise one ormore of amino acid residues 71, 72, 150-152, 187-192, 198-202, 212,214-217, 220-226, 243, 255-258, 317, 318, 347-351, 372, 373, 380, 382,and 383.

In still another embodiment, the invention provides an antibody whichspecifically binds PCSK9 comprising a VH complementary determiningregion one (CDR1) having the amino acid sequence shown in SEQ ID NO:8(SYYMH), a VH CDR2 having the amino acid sequence shown in SEQ ID NO:9(EISPFGGRTNYNEKFKS), and/or VH CDR3 having the amino acid sequence shownin SEQ ID NO:10 (ERPLYASDL), or a variant thereof having one or moreconservative amino acid substitutions in said sequences of CDR1, CDR2,and/or CDR3, wherein the variant retains essentially the same bindingspecificity as the CDR defined by said sequences. Preferably, thevariant comprises up to about ten amino acid substitutions and, morepreferably, up to about four amino acid substitutions.

The invention is further directed to an antibody comprising a VL CDR1having the amino acid sequence shown in SEQ ID NO:11 (RASQGISSALA), aCDR2 having the amino acid sequence shown in SEQ ID NO:12 (SASYRYT),and/or CDR3 having the amino acid sequence shown in SEQ ID NO:13(QQRYSLWRT), or a variant thereof having one or more conservative aminoacid substitutions in said sequences of CDR1, CDR2, and/or CDR3, whereinthe variant retains essentially the same binding specificity as the CDR1defined by said sequences. Preferably, the variant comprises up to aboutten amino acid substitutions and, more preferably, up to about fouramino acid substitutions.

In another embodiment, the invention provides an antibody comprisingspecific VL CDR1, CDR2, and/or CDR3 sequences, or a variant thereofhaving one or more conservative amino acid substitutions in CDR1, CDR2,and/or CDR3 and further comprising a VH complementary determining regionCDR1 having the amino acid sequence shown in SEQ ID NO:59, 60, or 8, aVH CDR2 having the amino acid sequence shown in SEQ ID NO:61 or 9,and/or VH CDR3 having the amino acid sequence shown in SEQ ID NO:10, ora variant thereof having one or more conservative amino acidsubstitutions in said sequences of CDR1, CDR2, and/or CDR3, wherein thevariant retains essentially the same binding specificity as the CDR1,CDR2, and/or CDR3 defined by said sequences. Preferably, the variantcomprises up to about twenty amino acid substitutions and, morepreferably, up to about eight amino acid substitutions. In anotherpreferred embodiment, the antibody of the invention has a variable heavychain sequence comprising or consisting of SEQ ID NO: 54 and a variablelight chain sequence comprising or consisting of SEQ ID NO: 53.

The invention also provides a humanized antibody comprising polypeptidesselected from the groups consisting of SEQ ID NO:14, SEQ ID NO:15, orboth SEQ ID NO:14 and SEQ ID NO:15, or a variant thereof having one ormore conservative amino acid substitutions in said sequences, whereinthe variant retains essentially the same binding specificity as theantibody defined by said sequence(s). It also includes an antibodylacking a terminal lysine on the heavy chain, as this is normally lostin a proportion of antibodies during manufacture.

Preferably, the variant comprises up to about twenty amino acidsubstitutions and more preferably, up to about eight amino acidsubstitutions. Preferably, the antibody further comprises animmunologically inert constant region, and/or the antibody has anisotype that is selected from the group consisting of IgG₂, IgG₄,IgG_(2Δa), IgG_(4Δb), IgG_(4Δc), IgG₄ S228P, IgG_(4Δb) S228P andIgG_(4Δc) S228P. In another preferred embodiment, the constant region isaglycosylated Fc.

In one embodiment, the invention provides a method for reducing a levelof LDL, LDL-cholesterol, or total cholesterol in blood, serum, or plasmaof a subject in need thereof, comprising administering to the subject atherapeutically effective amount of an antagonist of the invention.

In one embodiment, the invention provides a therapeutically effectiveamount of an antagonist of the invention for use in reducing a level ofLDL, LDL-cholesterol, or total cholesterol in blood, serum, or plasma ofa subject in need thereof. The invention further provides the use of atherapeutically effective amount of an antagonist of the invention inthe manufacture of a medicament for reducing a level of LDL,LDL-cholesterol, or total cholesterol in blood, serum, or plasma of asubject in need thereof.

In yet another embodiment, the invention provides a method of preparingan antibody which specifically binds PCSK9, which comprises: a)providing a PCSK9-negative host animal; b) immunizing saidPCSK9-negative host animal with PCSK9; and c) obtaining an antibody. Anantibody-producing cell, or an antibody-encoding nucleic acid from saidPCSK9-negative host animal, and preparing an antibody from saidantibody-producing cell or said antibody-encoding nucleic acid.

The invention also comprises a method for reducing the level of LDL inblood of a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of the antibody preparedaccording to the invention. The subject can be further treated byadministering a statin. In a preferred embodiment, the subject is ahuman subject.

In one embodiment, the antibody is administered in a formulation as asterile aqueous solution having a pH that ranges from about 5.0 to about6.5 and comprising from about 1 mg/ml to about 200 mg/ml of antibody,from about 1 millimolar to about 100 millimolar of histidine buffer,from about 0.01 mg/ml to about 10 mg/ml of polysorbate 80, from about100 millimolar to about 400 millimolar of trehalose, and from about 0.01millimolar to about 1.0 millimolar of disodium EDTA dihydrate.

In another embodiment, the invention provides a therapeuticallyeffective amount of the antibody prepared according to the invention foruse in reducing the level of LDL in blood of a subject in need thereof.The invention further provides the use of a therapeutically effectiveamount of the antibody prepared according to the invention in themanufacture of a medicament for reducing the level of LDL in blood of asubject in need thereof. The therapeutically effective amount canoptionally be combined with a therapeutically effective amount of astatin.

In another embodiment, the invention provides a hybridoma cell line thatproduces a PCSK9-specific antibody or an antigen-binding portionthereof, wherein the hybridoma cell line is selected from the groupconsisting of:

-   -   4A5 having an ATCC Accession No. of PTA-8985;    -   5A10 having an ATCC Accession No. of PTA-8986;    -   6F6 having an ATCC Accession No. of PTA-8984; and    -   7D4 having an ATCC Accession No. of PTA-8983.

In another embodiment, the invention provides cell line thatrecombinantly produces an antibody which specifically binds to PCSK9 andcomprises a heavy chain variable region (VH) complementary determiningregion one (CDR1) having the amino acid sequence shown in SEQ ID NO:8,59, or 60, a VH CDR2 having the amino acid sequence shown in SEQ ID NO:9or 61, and/or VH CDR3 having the amino acid sequence shown in SEQ IDNO:10, or a variant thereof having one or more conservative amino acidsubstitutions in CDR1, CDR2, and/or CDR3, and/or comprises a light chainvariable region (VL) CDR1 having the amino acid sequence shown in SEQ IDNO:11, a VL CDR2 having the amino acid sequence shown in SEQ ID NO:12,and/or VL CDR3 having the amino acid sequence shown in SEQ ID NO:13, ora variant thereof having one or more conservative amino acidsubstitutions in CDR1, CDR2, and/or CDR3. Preferably, the cell linerecombinantly produces an antibody comprising SEQ ID NO: 53 and/or 54,and, more preferably, SEQ ID NO: 14 and/or 15.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

FIG. 1 illustrates the effect of anti-PCSK9 antagonistic monoclonalantibodies 7D4.4, 4A5.G3, 6F6.G10.3 and 5A10.B8 on the ability of mousePCSK9 (A) and human PCSK9 (B) to down regulate LDLR in cultured Huh7cells. 6F6.G10.3 is a subclone of 6F6, 7D4.4 is a subclone of 7D4,4A5.G3 is a subclone of 4A5, and 5A10.B8 is a subclone of 5A10.

FIG. 2 illustrates the dose-response of anti-PCSK9 antagonist monoclonalantibodies 6F6.G10.3, 7D4.4, 4A5.G3, 5A10.B8, negative control antibody42H7, and PBS to block the binding of recombinant biotinylated humanPCSK9 (A) and mouse PCSK9 (B) to immobilized recombinant LDLRextracellular domain in vitro.

FIG. 3 illustrates the dose-response of anti-PCSK9 monoclonal antagonistantibodies 6F6.G10.3, 7D4.4, 4A5.G3 and 5A10.B8 to block binding ofrecombinant biotinylated human PCSK9 (30 nM) to Europium labeledrecombinant LDLR extracellular domain (10 nM) in solution at neutral pHin vitro.

FIGS. 4A and 4B illustrate comparative epitope binding of anti-PCSK9antibodies.

FIG. 5 illustrates Western blots of binding of anti-PCSK9 antibodies toserum PCSK9 from different species.

FIG. 6 illustrates the effect of anti-PCSK9 monoclonal antibody 7D4 onblood cholesterol levels in mice.

FIG. 7 illustrates (A) the effect of a partial antagonist polyclonalanti-PCSK9 mAb CRN6 on LDLR down regulation and (B) the lack of effecton cholesterol levels in mice.

FIGS. 8A and 8B illustrate the time course of the cholesterol loweringeffect obtained using anti-PCSK9 antagonist antibody 7D4 in mice.

FIGS. 9A, 9B and 9C illustrate the dose dependence of the anti-PCSK9antagonist mAb 7D4 on the reduction of serum total cholesterol, HDL andLDL in mice.

FIGS. 10A and 10B illustrate the dose dependence of the cholesterollowering effect of anti-PCSK9 antagonist antibody 5A10 in mice.

FIG. 11 illustrates the dose dependence of the cholesterol loweringeffect of anti-PCSK9 antagonist antibodies (A) 4A5 and (B) 6F6 in mice.

FIG. 12 depicts Western blots of anti-PCSK9 antagonist antibodies effecton liver LDLR levels.

FIG. 13 illustrates the lack of effect of anti-PCSK9 antagonist antibody4A5 in an LDLR−/− mouse model.

FIG. 14 illustrates the effect on total serum cholesterol of multipleadministrations of anti-PCSK9 antagonist antibodies in mice over alonger time course than seen with a single dose.

FIGS. 15A-H illustrate the time course of the effects of anti-PCSK9antagonistic antibody 7D4 on lipid parameters in a cynomolgus monkeymodel.

FIGS. 16A-D illustrate the dose- and time-response of anti-PCSK9antagonistic antibody 7D4 on serum cholesterol levels in the cynomolgusmonkey.

FIGS. 17A-D illustrate illustrates a comparison of anti-PCSK9antagonistic antibodies 4A5, 5A10, 6F6 and 7D4 on serum cholesterollevels in the cynomolgus monkey.

FIG. 18 illustrates the time course of the effect of anti-PCSK9antagonist antibody 7D4 on plasma cholesterol levels of cynomolgusmonkeys fed a 33.4% kcal fat diet supplemented with 0.1% cholesterol.

FIG. 19 illustrates the effect of L1L3 (humanized anti-PCSK9 monoclonalantibody) on down regulation of LDLR in Huh7 cells.

FIG. 20 illustrates the dose-response of L1L3 humanized antibody, themouse precursor 5A10, and negative control antibody 42H7 on blocking thebinding of recombinant biotinylated human PCSK9 (A and B) and mousePCSK9 (C and D) to immobilized recombinant LDLR extracellular domain invitro at pH 7.5 (A and C) and pH 5.3 (B and D).

FIG. 21 illustrates the effect on serum cholesterol of treatment of micewith 10 mg/kg L1L3.

FIG. 22 illustrates the effect of administration of 5A10 antibody orL1L3 to cynomolgus monkeys and measurement of changes in serum HDL (A)and serum LDL (B) as a function of time.

FIG. 23A depicts the crystal structure of the PCSK9 (light gray surfacerepresentation) bound to the L1L3 antibody (black cartoonrepresentation). FIG. 23B depicts the crystal structure of the PCSK9(light gray surface representation) bound to the EGF-like domain of theLDLR (black cartoon representation) (Kwon et al., PNAS, 105, 1820-1825,2008). FIG. 23C shows the surface area representation of PCSK9 with theL1L3 epitope shown in dark gray. FIG. 23D shows the surface arearepresentation of PCSK9 with the LDLR EGF-like domain epitope shown indark gray.

FIGS. 24 A-G depict the substitutions made in the CDRs of antibody 5A10in the course of affinity maturation and optimization and to achieveparticular properties. PCSK9 binding associated with antibodies havingthese CDR substitutions is also represented. The number following eachsequence is the SEQ ID NO designated for each sequence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to antibodies, peptides, and aptamers thatantagonize the function of extracellular PCSK9 including its interactionwith the LDLR. More specifically, the invention relates to methods ofmaking antagonist PCSK9 antibodies, peptides, and aptamers, compositionscomprising these antibodies, peptides, and/or aptamers, and methods ofusing these antibodies, peptides, and/or aptamers as a medicament. Theantagonist PCSK9 antibodies and peptides can be used to lower bloodLDL-cholesterol levels, and can be used in the prevention and/ortreatment of cholesterol and lipoprotein metabolism disorders, includingfamilial hypercholesterolemia, atherogenic dyslipidemia,atherosclerosis, and, more generally, CVD.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (AcademicPress, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti andJ.D. Capra, eds., Harwood Academic Publishers, 1995).

DEFINITIONS

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso fragments thereof (such as Fab, Fab′, F(ab′)₂, Fv), single chain(ScFv) and domain antibodies), and fusion proteins comprising anantibody portion, and any other modified configuration of theimmunoglobulin molecule that comprises an antigen recognition site. Anantibody includes an antibody of any class, such as IgG, IgA, or IgM (orsub-class thereof), and the antibody need not be of any particularclass. Depending on the antibody amino acid sequence of the constantdomain of its heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

As used herein, “monoclonal antibody” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler and Milstein, 1975, Nature 256:495, ormay be made by recombinant DNA methods such as described in U.S. Pat.No. 4,816,567. The monoclonal antibodies may also be isolated from phagelibraries generated using the techniques described in McCafferty et al.,1990, Nature 348:552-554, for example.

As used herein, “humanized” antibody refers to forms of non-human (e.g.,murine) antibodies that are chimeric immunoglobulins, immunoglobulinchains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) that contain minimalsequence derived from non-human immunoglobulin. Preferably, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat, or rabbit having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, the humanized antibody may compriseresidues that are found neither in the recipient antibody nor in theimported CDR or framework sequences, but are included to further refineand optimize antibody performance. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region or domain (Fc),typically that of a human immunoglobulin. Preferred are antibodieshaving Fc regions modified as described in WO 99/58572. Other forms ofhumanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDRH1, CDR H2, and/or CDR H3) which are altered with respect to theoriginal antibody, which are also termed one or more CDRs “derived from”one or more CDRs from the original antibody.

As used herein, “human antibody” means an antibody having an amino acidsequence corresponding to that of an antibody that can be produced by ahuman and/or which has been made using any of the techniques for makinghuman antibodies known to those skilled in the art or disclosed herein.This definition of a human antibody includes antibodies comprising atleast one human heavy chain polypeptide or at least one human lightchain polypeptide. One such example is an antibody comprising murinelight chain and human heavy chain polypeptides. Human antibodies can beproduced using various techniques known in the art. In one embodiment,the human antibody is selected from a phage library, where that phagelibrary expresses human antibodies (Vaughan et al., 1996, NatureBiotechnology, 14:309-314; Sheets et al., 1998, Proc. Natl. Acad. Sci.(USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381;Marks et al., 1991, J. Mol. Biol., 222:581). Human antibodies can alsobe made by immunization of animals into which human immunoglobulin locihave been transgenically introduced in place of the endogenous loci,e.g., mice in which the endogenous immunoglobulin genes have beenpartially or completely inactivated. This approach is described in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and5,661,016. Alternatively, the human antibody may be prepared byimmortalizing human B lymphocytes that produce an antibody directedagainst a target antigen (such B lymphocytes may be recovered from anindividual or may have been immunized in vitro). See, e.g., Cole et al.Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985;Boerner et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Pat. No.5,750,373.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. As known in the art, the variableregions of the heavy and light chain each consist of four frameworkregions (FR) connected by three complementarity determining regions(CDRs) that contain hypervariable regions. The CDRs in each chain areheld together in close proximity by the FRs and, with the CDRs from theother chain, contribute to the formation of the antigen-binding site ofantibodies. There are at least two techniques for determining CDRs: (1)an approach based on cross-species sequence variability (i.e., Kabat etal. Sequences of Proteins of Immunological Interest, (5th ed., 1991,National Institutes of Health, Bethesda Md.)); and (2) an approach basedon crystallographic studies of antigen-antibody complexes (Al-lazikaniet al, 1997, J. Molec. Biol. 273:927-948). As used herein, a CDR mayrefer to CDRs defined by either approach or by a combination of bothapproaches.

As known in the art a “constant region” of an antibody refers to theconstant region of the antibody light chain or the constant region ofthe antibody heavy chain, either alone or in combination.

As used herein, the term “PCSK9” refers to any form of PCSK9 andvariants thereof that retain at least part of the activity of PCSK9.Unless indicated differently, such as by specific reference to humanPCSK9, PCSK9 includes all mammalian species of native sequence PCSK9,e.g., human, canine, feline, equine, and bovine. One exemplary humanPCSK9 is found as Uniprot Accession Number Q8NBP7 (SEQ ID NO:16).

As used herein, a “PCSK9 antagonist” refers to an antibody, peptide, oraptamer that is able to inhibit PCSK9 biological activity and/ordownstream pathway(s) mediated by PCSK9 signaling, includingPCSK9-mediated down-regulation of the LDLR, and PCSK9-mediated decreasein LDL blood clearance. A PCSK9 antagonist antibody encompassesantibodies that block, antagonize, suppress or reduce (to any degreeincluding significantly) PCSK9 biological activity, including downstreampathways mediated by PCSK9 signaling, such as LDLR interaction and/orelicitation of a cellular response to PCSK9. For purpose of the presentinvention, it will be explicitly understood that the term “PCSK9antagonist antibody” encompasses all the previously identified terms,titles, and functional states and characteristics whereby the PCSK9itself, a PCSK9 biological activity (including but not limited to itsability to mediate any aspect of interaction with the LDLR, downregulation of LDLR, and decreased blood LDL clearance), or theconsequences of the biological activity, are substantially nullified,decreased, or neutralized in any meaningful degree. In some embodiments,a PCSK9 antagonist antibody binds PCSK9 and prevents interaction withthe LDLR. Examples of PCSK9 antagonist antibodies are provided herein.

As used herein a “full antagonist” is an antagonist which, at aneffective concentration, essentially completely blocks a measurableeffect of PCSK9. By a partial antagonist is meant an antagonist that iscapable of partially blocking a measurable effect, but that, even at ahighest concentration is not a full antagonist. By essentiallycompletely is meant at least about 80%, preferably, at least about 90%,more preferably, at least about 95%, and most preferably, at least about98% or 99% of the measurable effect is blocked. The relevant “measurableeffects” are described herein and include down regulation of LDLR by aPCSK9 antagonist as assayed in Huh7 cells in vitro, in vivo decrease inblood (or plasma) levels of total cholesterol, and in vivo decrease inLDL levels in blood (or plasma).

As used herein, the term “clinically meaningful” means at least a 15%reduction in blood LDL-cholesterol levels in humans or at least a 15%reduction in total blood cholesterol in mice. It is clear thatmeasurements in plasma or serum can serve as surrogates for measurementof levels in blood.

As used herein, the term “PCSK9 antagonist peptide” or “PCSK9 antagonistaptamer” includes any conventional peptide or polypeptide or aptamerthat blocks, antagonizes, suppresses or reduces (to any degree includingsignificantly) PCSK9 biological activity, including downstream pathwaysmediated by PCSK9 signaling, such as LDLR interaction and/or elicitationof a cellular response to PCSK9. PCSK9 antagonist peptides orpolypeptides include Fc fusions comprising the LDLR and soluble portionsof the LDLR, or mutants thereof with higher affinity to PCSK9.

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to chains of amino acids of anylength, preferably, relatively short (e.g., 10-100 amino acids). Thechain may be linear or branched, it may comprise modified amino acids,and/or may be interrupted by non-amino acids. The terms also encompassan amino acid chain that has been modified naturally or by intervention;for example, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications known in the art. It is understood thatthe polypeptides can occur as single chains or associated chains.

As known in the art, “polynucleotide,” or “nucleic acid,” as usedinterchangeably herein, refer to chains of nucleotides of any length,and include DNA and RNA. The nucleotides can be deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, and/or their analogs, orany substrate that can be incorporated into a chain by DNA or RNApolymerase. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thechain. The sequence of nucleotides may be interrupted by non-nucleotidecomponents. A polynucleotide may be further modified afterpolymerization, such as by conjugation with a labeling component. Othertypes of modifications include, for example, “caps”, substitution of oneor more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide(s).Further, any of the hydroxyl groups ordinarily present in the sugars maybe replaced, for example, by phosphonate groups, phosphate groups,protected by standard protecting groups, or activated to prepareadditional linkages to additional nucleotides, or may be conjugated tosolid supports. The 5′ and 3′ terminal OH can be phosphorylated orsubstituted with amines or organic capping group moieties of from 1 to20 carbon atoms. Other hydroxyls may also be derivatized to standardprotecting groups. Polynucleotides can also contain analogous forms ofribose or deoxyribose sugars that are generally known in the art,including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or2′-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomericsugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranosesugars, furanose sugars, sedoheptuloses, acyclic analogs and abasicnucleoside analogs such as methyl riboside. One or more phosphodiesterlinkages may be replaced by alternative linking groups. Thesealternative linking groups include, but are not limited to, embodimentswherein phosphate is replaced by P(O)S(“thioate”), P(S)S (“dithioate”),(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂ (“formacetal”), in whicheach R or R′ is independently H or substituted or unsubstituted alkyl(1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl,cycloalkyl, cycloalkenyl or araldyl. Not all linkages in apolynucleotide need be identical. The preceding description applies toall polynucleotides referred to herein, including RNA and DNA.

A “PCSK9 antagonist aptamer,” which comprises a nucleic acid or proteinsequence, is, for example, selected from a large pool of randomsequences and specifically binds PCSK9. The nucleic acid of the aptameris double-stranded DNA or single-strand RNA. Nucleic acid aptamers caninclude modified bases or functional groups, including but not limitedto 2′-fluorine nucleotides and 2′-O-methyl nucleotides. Aptamers caninclude hydrophilic polymers, for example, polyethylene glycol. Aptamersmay be made by methods known in the art and selected for PCSK9antagonist activity by routine modification of the methods disclosed inthe Examples.

As used herein, an antibody, peptide, or aptamer “interacts with” PCSK9when the equilibrium dissociation constant is equal to or less than 20nM, preferably less than about 6 nM, more preferably less than about 1nM, most preferably less than about 0.2 nM, as measured by the methodsdisclosed herein in Example 2.

An epitope that “preferentially binds” or “specifically binds” (usedinterchangeably herein) to an antibody or a polypeptide is a term wellunderstood in the art, and methods to determine such specific orpreferential binding are also well known in the art. A molecule is saidto exhibit “specific binding” or “preferential binding” if it reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular cell or substance than it doeswith alternative cells or substances. An antibody “specifically binds”or “preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. For example, an antibody that specifically orpreferentially binds to a PCSK9 epitope is an antibody that binds thisepitope with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other PCSK9 epitopes or non-PCSK9epitopes. It is also understood by reading this definition that, forexample, an antibody (or moiety or epitope) that specifically orpreferentially binds to a first target may or may not specifically orpreferentially bind to a second target. As such, “specific binding” or“preferential binding” does not necessarily require (although it caninclude) exclusive binding. Generally, but not necessarily, reference tobinding means preferential binding.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), more preferably, at least90% pure, more preferably, at least 95% pure, yet more preferably, atleast 98% pure, and most preferably, at least 99% pure.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of this invention.

As known in the art, the term “Fc region” is used to define a C-terminalregion of an immunoglobulin heavy chain. The “Fc region” may be a nativesequence Fc region or a variant Fc region. Although the boundaries ofthe Fc region of an immunoglobulin heavy chain might vary, the human IgGheavy chain Fc region is usually defined to stretch from an amino acidresidue at position Cys226, or from Pro230, to the carboxyl-terminusthereof. The numbering of the residues in the Fc region is that of theEU index as in Kabat. Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991. The Fc region of animmunoglobulin generally comprises two constant domains, CH2 and CH3.

As used in the art, “Fc receptor” and “FcR” describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. FcRs are reviewed in Ravetch and Kinet,1991, Ann. Rev. Immunol., 9:457-92; Capel et al., 1994, Immunomethods,4:25-34; and de Haas et al., 1995, J. Lab. Clin. Med., 126:330-41. “FcR”also includes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., 1976 J. Immunol.,117:587; and Kim et al., 1994, J. Immunol., 24:249).

The term “compete”, as used herein with regard to an antibody, meansthat a first antibody, or an antigen-binding portion thereof, binds toan epitope in a manner sufficiently similar to the binding of a secondantibody, or an antigen-binding portion thereof, such that the result ofbinding of the first antibody with its cognate epitope is detectablydecreased in the presence of the second antibody compared to the bindingof the first antibody in the absence of the second antibody. Thealternative, where the binding of the second antibody to its epitope isalso detectably decreased in the presence of the first antibody, can,but need not be the case. That is, a first antibody can inhibit thebinding of a second antibody to its epitope without that second antibodyinhibiting the binding of the first antibody to its respective epitope.However, where each antibody detectably inhibits the binding of theother antibody with its cognate epitope or ligand, whether to the same,greater, or lesser extent, the antibodies are said to “cross-compete”with each other for binding of their respective epitope(s). Bothcompeting and cross-competing antibodies are encompassed by the presentinvention. Regardless of the mechanism by which such competition orcross-competition occurs (e.g., steric hindrance, conformational change,or binding to a common epitope, or portion thereof), the skilled artisanwould appreciate, based upon the teachings provided herein, that suchcompeting and/or cross-competing antibodies are encompassed and can beuseful for the methods disclosed herein.

By an antibody with an epitope that “overlaps” with another (second)epitope or with a surface on PCSK9 that interacts with the EGF-likedomain of the LDLR is meant the sharing of space in terms of the PCSK9residues that are interacted with. To calculate the percent of overlap,for example, the percent overlap of the claimed antibody's PCSK9 epitopewith the surface of PCSK9 which interacts with the EGF-like domain ofthe LDLR, the surface area of PCSK9 buried when in complex with the LDLRis calculated on a per-residue basis. The buried area is also calculatedfor these residues in the PCSK9:antibody complex. To prevent more than100% possible overlap, surface area for residues that have higher buriedsurface area in the PCSK9:antibody complex than in LDLR:PCSK9 complex isset to values from the LDLR:PCSK9 complex (100%). Percent surfaceoverlap is calculated by summing over all of the LDLR:PCSK9 interactingresidues and is weighted by the interaction area.

A “functional Fc region” possesses at least one effector function of anative sequence Fc region. Exemplary “effector functions” include Clqbinding; complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity; phagocytosis;down-regulation of cell surface receptors (e.g., B cell receptor), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g., an antibody variable domain) and can beassessed using various assays known in the art for evaluating suchantibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” comprises an amino acid sequence which differs from that of anative sequence Fc region by virtue of at least one amino acidmodification, yet retains at least one effector function of the nativesequence Fc region. Preferably, the variant Fc region has at least oneamino acid substitution compared to a native sequence Fc region or tothe Fc region of a parent polypeptide, e.g., from about one to about tenamino acid substitutions, and preferably, from about one to about fiveamino acid substitutions in a native sequence Fc region or in the Fcregion of the parent polypeptide. The variant Fc region herein willpreferably possess at least about 80% sequence identity with a nativesequence Fc region and/or with an Fc region of a parent polypeptide, andmost preferably, at least about 90% sequence identity therewith, morepreferably, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99% sequence identity therewith.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, one or more ofthe following: enhancement of LDL clearance and reducing incidence oramelioration of aberrant cholesterol and/or lipoprotein levels resultingfrom metabolic and/or eating disorders, or including familialhypercholesterolemia, atherogenic dyslipidemia, atherosclerosis, and,more generally, cardiovascular disease (CVD).

“Reducing incidence” means any of reducing severity (which can includereducing need for and/or amount of (e.g., exposure to) other drugsand/or therapies generally used for this condition. As is understood bythose skilled in the art, individuals may vary in terms of theirresponse to treatment, and, as such, for example, a “method of reducingincidence” reflects administering the PCSK9 antagonist antibody,peptide, or aptamer based on a reasonable expectation that suchadministration may likely cause such a reduction in incidence in thatparticular individual.

“Ameliorating” means a lessening or improvement of one or more symptomsas compared to not administering a PCSK9 antagonist antibody, peptide,or aptamer. “Ameliorating” also includes shortening or reduction induration of a symptom.

As used herein, an “effective dosage” or “effective amount” of drug,compound, or pharmaceutical composition is an amount sufficient toeffect any one or more beneficial or desired results. For prophylacticuse, beneficial or desired results include eliminating or reducing therisk, lessening the severity, or delaying the outset of the disease,including biochemical, histological and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. For therapeutic use,beneficial or desired results include clinical results such as reducinghypercholesterolemia or one or more symptoms of dyslipidemia,atherosclerosis, CVD, or coronary heart disease, decreasing the dose ofother medications required to treat the disease, enhancing the effect ofanother medication, and/or delaying the progression of the disease ofpatients. An effective dosage can be administered in one or moreadministrations. For purposes of this invention, an effective dosage ofdrug, compound, or pharmaceutical composition is an amount sufficient toaccomplish prophylactic or therapeutic treatment either directly orindirectly. As is understood in the clinical context, an effectivedosage of a drug, compound, or pharmaceutical composition may or may notbe achieved in conjunction with another drug, compound, orpharmaceutical composition. Thus, an “effective dosage” may beconsidered in the context of administering one or more therapeuticagents, and a single agent may be considered to be given in an effectiveamount if, in conjunction with one or more other agents, a desirableresult may be or is achieved.

An “individual” or a “subject” is a mammal, more preferably, a human.Mammals also include, but are not limited to, farm animals, sportanimals, pets, primates, horses, dogs, cats, mice and rats.

As used herein, “vector” means a construct, which is capable ofdelivering, and, preferably, expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

As used herein, “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid. An expressioncontrol sequence can be a promoter, such as a constitutive or aninducible promoter, or an enhancer. The expression control sequence isoperably linked to the nucleic acid sequence to be transcribed.

As used herein, “pharmaceutically acceptable carrier” or “pharmaceuticalacceptable excipient” includes any material which, when combined with anactive ingredient, allows the ingredient to retain biological activityand is non-reactive with the subject's immune system. Examples include,but are not limited to, any of the standard pharmaceutical carriers suchas a phosphate buffered saline solution, water, emulsions such asoil/water emulsion, and various types of wetting agents. Preferreddiluents for aerosol or parenteral administration are phosphate bufferedsaline (PBS) or normal (0.9%) saline. Compositions comprising suchcarriers are formulated by well known conventional methods (see, forexample, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro,ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Scienceand Practice of Pharmacy, 20th Ed., Mack Publishing, 2000).

The term “k_(on)”, as used herein, refers to the rate constant forassociation of an antibody to an antigen. Specifically, the rateconstants (k_(m) and k_(off)) and equilibrium dissociation constants aremeasured using Fab antibody fragments (i.e., univalent) and PCSK9.

The term “k_(off)”, as used herein, refers to the rate constant fordissociation of an antibody from the antibody/antigen complex.

The term “K_(D)”, as used herein, refers to the equilibrium dissociationconstant of an antibody-antigen interaction.

A. Methods for Preventing or Treating Disorders Associated withHypercholesterolemia

In one aspect, the invention provides a method for treating orpreventing hypercholesterolemia, and/or at least one symptom ofdyslipidemia, atherosclerosis, CVD or coronary heart disease, in anindividual comprising administering to the individual an effectiveamount of a PCSK9 antagonist antibody or peptide or aptamer thatantagonizes circulating PCSK9.

In a further aspect, the invention provides an effective amount of aPCSK9 antagonist antibody, peptide, or aptamer that antagonizescirculating PCSK9 for use in treating or preventinghypercholesterolemia, and/or at least one symptom of dyslipidemia,atherosclerosis, CVD or coronary heart disease, in an individual. Theinvention further provides the use of an effective amount of a PCSK9antagonist antibody, peptide, or aptamer that antagonizes extracellularor circulating PCSK9 in the manufacture of a medicament for treating orpreventing hypercholesterolemia, and/or at least one symptom ofdyslipidemia, atherosclerosis, CVD or coronary heart disease, in anindividual.

Advantageously, therapeutic administration of the antibody, peptide, oraptamer results in lower blood cholesterol and/or lower blood LDL.Preferably, blood cholesterol and/or blood LDL is at least about 10% or15% lower than before administration. More preferably, blood cholesteroland/or blood LDL is at least about 20% lower than before administrationof the antibody. Yet more preferably, blood cholesterol and/or blood LDLis at least 30% lower than before administration of the antibody.Advantageously, blood cholesterol and/or blood LDL is at least 40% lowerthan before administration of the antibody. More advantageously, bloodcholesterol and/or blood LDL is at least 50% lower than beforeadministration of the antibody. Very preferably, blood cholesteroland/or blood LDL is at least 60% lower than before administration of theantibody. Most preferably, blood cholesterol and/or blood LDL is atleast 70% lower than before administration of the antibody.

With respect to all methods described herein, reference to PCSK9antagonist antibodies, peptides, and aptamers also include compositionscomprising one or more additional agents. These compositions may furthercomprise suitable excipients, such as pharmaceutically acceptableexcipients including buffers, which are well known in the art. Thepresent invention can be used alone or in combination with otherconventional methods of treatment.

The PCSK9 antagonist antibody, peptide, or aptamer can be administeredto an individual via any suitable route. It should be apparent to aperson skilled in the art that the examples described herein are notintended to be limiting but to be illustrative of the techniquesavailable. Accordingly, in some embodiments, the PCSK9 antagonistantibody, peptide, or aptamer is administered to an individual in accordwith known methods, such as intravenous administration, e.g., as a bolusor by continuous infusion over a period of time, by intramuscular,intraperitoneal, intracerebrospinal, transdermal, subcutaneous,intra-articular, sublingually, intrasynovial, via insufflation,intrathecal, oral, inhalation or topical routes. Administration can besystemic, e.g., intravenous administration, or localized. Commerciallyavailable nebulizers for liquid formulations, including jet nebulizersand ultrasonic nebulizers are useful for administration. Liquidformulations can be directly nebulized and lyophilized powder can benebulized after reconstitution. Alternatively, PCSK9 antagonistantibody, peptide, or aptamer can be aerosolized using a fluorocarbonformulation and a metered dose inhaler, or inhaled as a lyophilized andmilled powder.

In one embodiment, a PCSK9 antagonist antibody, peptide, or aptamer isadministered via site-specific or targeted local delivery techniques.Examples of site-specific or targeted local delivery techniques includevarious implantable depot sources of the PCSK9 antagonist antibody,peptide, or aptamer or local delivery catheters, such as infusioncatheters, indwelling catheters, or needle catheters, synthetic grafts,adventitial wraps, shunts and stents or other implantable devices, sitespecific carriers, direct injection, or direct application. See, e.g.,PCT Publ. No. WO 00/53211 and U.S. Pat. No. 5,981,568.

Various formulations of a PCSK9 antagonist antibody, peptide, or aptamermay be used for administration. In some embodiments, the PCSK9antagonist antibody, peptide, or aptamer may be administered neat. Insome embodiments, PCSK9 antagonist antibody, peptide, or aptamer and apharmaceutically acceptable excipient may be in various formulations.Pharmaceutically acceptable excipients are known in the art, and arerelatively inert substances that facilitate administration of apharmacologically effective substance. For example, an excipient cangive form or consistency, or act as a diluent. Suitable excipientsinclude but are not limited to stabilizing agents, wetting andemulsifying agents, salts for varying osmolarity, encapsulating agents,buffers, and skin penetration enhancers. Excipients as well asformulations for parenteral and nonparenteral drug delivery are setforth in Remington, The Science and Practice of Pharmacy, 20th Ed., MackPublishing (2000).

These agents can be combined with pharmaceutically acceptable vehiclessuch as saline, Ringer's solution, dextrose solution, and the like. Theparticular dosage regimen, i.e., dose, timing and repetition, willdepend on the particular individual and that individual's medicalhistory.

PCSK9 antibodies can also be administered via inhalation, as describedherein. Generally, for administration of PCSK9 antibodies, an initialcandidate dosage can be about 2 mg/kg. For the purpose of the presentinvention, a typical daily dosage might range from about any of about 3μg/kg to 30 μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg, to 100 mg/kg ormore, depending on the factors mentioned above. For example, dosage ofabout 1 mg/kg, about 2.5 mg/kg, about 5 mg/kg, about 10 mg/kg, and about25 mg/kg may be used. For repeated administrations over several days orlonger, depending on the condition, the treatment is sustained until adesired suppression of symptoms occurs or until sufficient therapeuticlevels are achieved, for example, to reduce blood LDL levels. Anexemplary dosing regimen comprises administering an initial dose ofabout 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg ofthe PCSK9 antibody, or followed by a maintenance dose of about 1 mg/kgevery other week. However, other dosage regimens may be useful,depending on the pattern of pharmacokinetic decay that the practitionerwishes to achieve. For example, in some embodiments, dosing from one tofour times a week is contemplated. In other embodiments dosing once amonth or once every other month or every three months is contemplated.The progress of this therapy is easily monitored by conventionaltechniques and assays. The dosing regimen (including the PCSK9antagonist(s) used) can vary over time.

For the purpose of the present invention, the appropriate dosage of aPCSK9 antagonist antibody, peptide, or aptamer will depend on the PCSK9antagonist antibody, peptide, or aptamer (or compositions thereof)employed, the type and severity of symptoms to be treated, whether theagent is administered for preventive or therapeutic purposes, previoustherapy, the patient's clinical history and response to the agent, thepatient's blood PCSK9 levels, the patient's synthesis and clearance ratefor PCSK9, the patient's clearance rate for the administered agent, andthe discretion of the attending physician. Typically the clinician willadminister a PCSK9 antagonist antibody, peptide, or aptamer until adosage is reached that achieves the desired result. Dose and/orfrequency can vary over course of treatment. Empirical considerations,such as the half-life, generally will contribute to the determination ofthe dosage. For example, antibodies that are compatible with the humanimmune system, such as humanized antibodies or fully human antibodies,may be used to prolong half-life of the antibody and to prevent theantibody being attacked by the host's immune system. Frequency ofadministration may be determined and adjusted over the course oftherapy, and is generally, but not necessarily, based on treatmentand/or suppression and/or amelioration and/or delay of symptoms, e.g.,hypercholesterolemia. Alternatively, sustained continuous releaseformulations of PCSK9 antagonist antibodies may be appropriate. Variousformulations and devices for achieving sustained release are known inthe art.

In one embodiment, dosages for an antagonist antibody, peptide, oraptamer may be determined empirically in individuals who have been givenone or more administration(s) of an antagonist antibody, peptide, oraptamer. Individuals are given incremental dosages of a PCSK9 antagonistantibody, peptide, or aptamer. To assess efficacy, an indicator of thedisease can be followed.

Administration of a PCSK9 antagonist antibody, peptide, or aptamer inaccordance with the method in the present invention can be continuous orintermittent, depending, for example, upon the recipient's physiologicalcondition, whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of a PCSK9 antagonist antibody, peptide, or aptamer maybe essentially continuous over a preselected period of time or may be ina series of spaced doses.

In some embodiments, more than one antagonist antibody, peptide, oraptamer may be present. At least one, at least two, at least three, atleast four, at least five different, or more antagonist antibodiesand/or peptides can be present. Generally, those PCSK9 antagonistantibodies or peptides may have complementary activities that do notadversely affect each other. A PCSK9 antagonist antibody, peptide, oraptamer can also be used in conjunction with other PCSK9 antagonists orPCSK9 receptor antagonists. For example, one or more of the followingPCSK9 antagonists may be used: an antisense molecule directed to a PCSK9(including an anti-sense molecule directed to a nucleic acid encodingPCSK9), a PCSK9 inhibitory compound, and a PCSK9 structural analog. APCSK9 antagonist antibody, peptide, or aptamer can also be used inconjunction with other agents that serve to enhance and/or complementthe effectiveness of the agents.

Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and may comprisebuffers such as phosphate, citrate, and other organic acids; salts suchas sodium chloride; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl orpropyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

Liposomes containing the PCSK9 antagonist antibody, peptide, or aptamerare prepared by methods known in the art, such as described in Epstein,et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688; Hwang, et al., 1980,Proc. Natl. Acad. Sci. USA 77:4030; and U.S. Pat. Nos. 4,485,045 and4,544,545. Liposomes with enhanced circulation time are disclosed inU.S. Pat. No. 5,013,556. Particularly useful liposomes can be generatedby the reverse phase evaporation method with a lipid compositioncomprising phosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington, The Science and Practice of Pharmacy, 20th Ed., MackPublishing (2000).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or ‘poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by, for example, filtration through sterilefiltration membranes. Therapeutic PCSK9 antagonist antibody, peptide, oraptamer compositions are generally placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

Suitable emulsions may be prepared using commercially available fatemulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ andLipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g., egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%. The fat emulsion can comprise fat dropletsbetween 0.1 and 1.0 μm, particularly 0.1 and 0.5 μm, and have a pH inthe range of 5.5 to 8.0.

The emulsion compositions can be those prepared by mixing a PCSK9antagonist antibody, peptide, or aptamer with Intralipid™ or thecomponents thereof (soybean oil, egg phospholipids, glycerol and water).

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as set outabove. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulised by use of gases. Nebulised solutions may be breatheddirectly from the nebulising device or the nebulising device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner.

B. PCSK9 Antagonists

The methods of the invention use a PCSK9 antagonist antibody, peptide,or aptamer, which refers to any peptide or nucleic acid molecule thatblocks, suppresses or reduces (including significantly reduces) PCSK9biological activity, including downstream pathways mediated by PCSK9signaling, such as elicitation of a cellular response to PCSK9.

A PCSK9 antagonist antibody, peptide, or aptamer should exhibit any oneor more of the following characteristics: (a) bind to PCSK9; (b) blockPCSK9 interaction with the LDLR; (c) block or decrease PCSK9-mediateddown-regulation of the LDLR; (d) inhibit the PCSK9-mediated decrease inLDL blood clearance, (e) increase LDL clearance in media by culturedhepatocytes, (f) increase blood LDL clearance by the liver in vivo, (g)sensitize to statins, and (h) block PCSK9 interaction with other yet tobe identified factors.

For purposes of this invention, the antibody, peptide, or aptamerpreferably reacts with PCSK9 in a manner that inhibits PCSK9 signalingfunction and LDLR interaction. In some embodiments, the PCSK9 antagonistantibody specifically recognizes primate PCSK9. In some embodiments, thePCSK9 antagonist antibody binds primate and rodent PCSK9.

The antibodies useful in the present invention can encompass monoclonalantibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab′,F(ab′)2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies,heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusionproteins comprising an antibody portion (e.g., a domain antibody), humanantibodies, humanized antibodies, and any other modified configurationof the immunoglobulin molecule that comprises an antigen recognitionsite of the required specificity, including glycosylation variants ofantibodies, amino acid sequence variants of antibodies, and covalentlymodified antibodies. The antibodies may be murine, rat, human, or anyother origin (including chimeric or humanized antibodies).

In some embodiments, the PCSK9 antagonist antibody is a monoclonalantibody. The PCSK9 antagonist antibody can also be humanized. In otherembodiments, the antibody is human.

In some embodiments, the antibody comprises a modified constant region,such as a constant region that is immunologically inert, that is, havinga reduced potential for provoking an immune response. In someembodiments, the constant region is modified as described in Eur. J.Immunol., 1999, 29:2613-2624; PCT Publ. No. WO99/58572; and/or UK PatentApplication No. 9809951.8. The Fc can be human IgG₂ or human IgG₄. TheFc can be human IgG₂ containing the mutation A330P331 to S330S331(IgG_(2Δa)), in which the amino acid residues are numbered withreference to the wild type IgG2 sequence. Eur. J. Immunol., 1999,29:2613-2624. In some embodiments, the antibody comprises a constantregion of IgG₄ comprising the following mutations (Armour et al., 2003,Molecular Immunology 40 585-593): E233F234L235 to P233V234A235(IgG_(4Δc)), in which the numbering is with reference to wild type IgG4.In yet another embodiment, the Fc is human IgG₄ E233F234L235 toP233V234A235 with deletion G236 (IgG_(4Δb)). In another embodiment theFc is any human IgG₄ Fc (IgG₄, IgG_(4Δb) or IgG_(4Δc)) containing hingestabilizing mutation S228 to P228 (Aalberse et al., 2002, Immunology105, 9-19). In another embodiment, the Fc can be aglycosylated Fc.

In some embodiments, the constant region is aglycosylated by mutatingthe oligosaccharide attachment residue (such as Asn297) and/or flankingresidues that are part of the glycosylation recognition sequence in theconstant region. In some embodiments, the constant region isaglycosylated for N-linked glycosylation enzymatically. The constantregion may be aglycosylated for N-linked glycosylation enzymatically orby expression in a glycosylation deficient host cell.

The binding affinity (K_(D)) of a PCSK9 antagonist antibody to PCSK9(such as human PCSK9)) can be about 0.002 to about 200 nM. In someembodiments, the binding affinity is any of about 200 nM, about 100 nM,about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, about60 pM, about 50 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM,or about 2 pM. In some embodiments, the binding affinity is less thanany of about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 10nM, about 1 nM, about 500 pM, about 100 pM, about 50 pM, about 20 pM,about 10 pM, about 5 pM, or about 2 pM.

One way of determining binding affinity of antibodies to PCSK9 is bymeasuring binding affinity of monofunctional Fab fragments of theantibody. To obtain monofunctional Fab fragments, an antibody (forexample, IgG) can be cleaved with papain or expressed recombinantly. Theaffinity of a PCSK9 Fab fragment of an antibody can be determined bysurface plasmon resonance (Biacore3000™ surface plasmon resonance (SPR)system, Biacore, INC, Piscataway N.J.) equipped with pre-immobilizedstreptavidin sensor chips (SA) using HBS-EP running buffer (0.01M HEPES,pH 7.4, 0.15 NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20). Biotinylatedhuman PCSK9 (or any other PCSK9) can be diluted into HBS-EP buffer to aconcentration of less than 0.5 μg/mL and injected across the individualchip channels using variable contact times, to achieve two ranges ofantigen density, either 50-200 response units (RU) for detailed kineticstudies or 800-1,000 RU for screening assays. Regeneration studies haveshown that 25 mM NaOH in 25% v/v ethanol effectively removes the boundFab while keeping the activity of PCSK9 on the chip for over 200injections. Typically, serial dilutions (spanning concentrations of0.1-10× estimated K_(D)) of purified Fab samples are injected for 1 minat 100 μL/minute and dissociation times of up to 2 hours are allowed.The concentrations of the Fab proteins are determined by ELISA and/orSDS-PAGE electrophoresis using a Fab of known concentration (asdetermined by amino acid analysis) as a standard. Kinetic associationrates (k_(on)) and dissociation rates (k_(off)) are obtainedsimultaneously by fitting the data globally to a 1:1 Langmuir bindingmodel (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. MethodsEnzymology 6. 99-110) using the BIAevaluation program. Equilibriumdissociation constant (K_(D)) values are calculated as k_(off)/k_(on).This protocol is suitable for use in determining binding affinity of anantibody to any PCSK9, including human PCSK9, PCSK9 of another mammalian(such as mouse PCSK9, rat PCSK9, primate PCSK9), as well as differentforms of PCSK9 (such as α and β form). Binding affinity of an antibodyis generally measured at 25° C., but can also be measured at 37° C.

The PCSK9 antagonist antibodies may be made by any method known in theart including the method as provided in Example 1. The route andschedule of immunization of the host animal are generally in keepingwith established and conventional techniques for antibody stimulationand production, as further described herein. General techniques forproduction of human and mouse antibodies are known in the art and/or aredescribed herein. A currently preferred method of making the antibodiescomprises the immunization of PCSK9⁻ knockout (PCSK9−/−) animals asdisclosed herein.

It is contemplated that any mammalian subject including humans orantibody producing cells therefrom can be manipulated to serve as thebasis for production of mammalian, including human, hybridoma celllines. Typically, the host animal is inoculated intraperitoneally,intramuscularly, orally, subcutaneously, intraplantar, and/orintradermally with an amount of immunogen, including as describedherein.

Hybridomas can be prepared from the lymphocytes and immortalized myelomacells using the general somatic cell hybridization technique of Kohler,B. and Milstein, C., 1975, Nature 256:495-497 or as modified by Buck, D.W., et al., 1982, In Vitro, 18:377-381. Available myeloma lines,including but not limited to X63-Ag8.653 and those from the SalkInstitute, Cell Distribution Center, San Diego, Calif., USA, may be usedin the hybridization. Generally, the technique involves fusing myelomacells and lymphoid cells using a fusogen such as polyethylene glycol, orby electrical means well known to those skilled in the art. After thefusion, the cells are separated from the fusion medium and grown in aselective growth medium, such as hypoxanthine-aminopterin-thymidine(HAT) medium, to eliminate unhybridized parent cells. Any of the mediadescribed herein, supplemented with or without serum, can be used forculturing hybridomas that secrete monoclonal antibodies. As anotheralternative to the cell fusion technique, EBV immortalized B cells maybe used to produce the PCSK9 monoclonal antibodies of the subjectinvention. The hybridomas are expanded and subcloned, if desired, andsupernatants are assayed for anti-immunogen activity by conventionalimmunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, orfluorescence immunoassay).

Hybridomas that may be used as a source of antibodies encompass allderivatives, progeny cells of the parent hybridomas that producemonoclonal antibodies specific for PCSK9, or a portion thereof.

Hybridomas that produce such antibodies may be grown in vitro or in vivousing known procedures. The monoclonal antibodies may be isolated fromthe culture media or body fluids, by conventional immunoglobulinpurification procedures such as ammonium sulfate precipitation, gelelectrophoresis, dialysis, chromatography, and ultrafiltration, ifdesired. Undesired activity, if present, can be removed, for example, byrunning the preparation over adsorbents made of the immunogen attachedto a solid phase and eluting or releasing the desired antibodies off theimmunogen. Immunization of a host animal with a human PCSK9, or afragment containing the target amino acid sequence conjugated to aprotein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups, can yield a population ofantibodies (e.g., monoclonal antibodies).

If desired, the PCSK9 antagonist antibody (monoclonal or polyclonal) ofinterest may be sequenced and the polynucleotide sequence may then becloned into a vector for expression or propagation. The sequenceencoding the antibody of interest may be maintained in vector in a hostcell and the host cell can then be expanded and frozen for future use.Production of recombinant monoclonal antibodies in cell culture can becarried out through cloning of antibody genes from B cells by meansknown in the art. See, e.g., Tiller et al., 2008, J. Immunol. Methods329, 112; U.S. Pat. No. 7,314,622.

In an alternative, the polynucleotide sequence may be used for geneticmanipulation to “humanize” the antibody or to improve the affinity, orother characteristics of the antibody. For example, the constant regionmay be engineered to more nearly resemble human constant regions toavoid immune response if the antibody is used in clinical trials andtreatments in humans. It may be desirable to genetically manipulate theantibody sequence to obtain greater affinity to PCSK9 and greaterefficacy in inhibiting PCSK9. It will be apparent to one of skill in theart that one or more polynucleotide changes can be made to the PCSK9antagonist antibody and still maintain its binding ability to PCSK9.

There are four general steps to humanize a monoclonal antibody. Theseare: (1) determining the nucleotide and predicted amino acid sequence ofthe starting antibody light and heavy variable domains; (2) designingthe humanized antibody, i.e., deciding which antibody framework regionto use during the humanizing process; (3) the actual humanizingmethodologies/techniques; and (4) the transfection and expression of thehumanized antibody. See, for example, U.S. Pat. Nos. 4,816,567;5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762;5,585,089; and 6,180,370.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent or modified rodent V regionsand their associated CDRs fused to human constant domains. See, forexample, Winter et al., 1991, Nature 349:293-299; Lobuglio et al., 1989,Proc. Nat. Acad. Sci. USA 86:4220-4224; Shaw et al., 1987, J. Immunol.138:4534-4538; and Brown et al., 1987, Cancer Res. 47:3577-3583. Otherreferences describe rodent CDRs grafted into a human supportingframework region (FR) prior to fusion with an appropriate human antibodyconstant domain. See, for example, Riechmann et al., 1988, Nature332:323-327; Verhoeyen et al., 1988, Science 239:1534-1536; and Jones etal., 1986, Nature 321:522-525. Another reference describes rodent CDRssupported by recombinantly engineered rodent framework regions. See, forexample, European Patent Publ. No. 0519596. These “humanized” moleculesare designed to minimize unwanted immunological response toward rodentanti-human antibody molecules which limits the duration andeffectiveness of therapeutic applications of those moieties in humanrecipients. For example, the antibody constant region can be engineeredsuch that it is immunologically inert (e.g., does not trigger complementlysis). See, e.g., PCT Publ. No. WO99/58572; UK Patent Application No.9809951.8. Other methods of humanizing antibodies that may also beutilized are disclosed by Daugherty et al., 1991, Nucl. Acids Res.19:2471-2476 and in U.S. Pat. Nos. 6,180,377; 6,054,297; 5,997,867;5,866,692; 6,210,671; and 6,350,861; and in PCT Publ. No. WO 01/27160.

In yet another alternative, fully human antibodies may be obtained byusing commercially available mice that have been engineered to expressspecific human immunoglobulin proteins. Transgenic animals that aredesigned to produce a more desirable or more robust immune response mayalso be used for generation of humanized or human antibodies. Examplesof such technology are Xenomouse™ from Abgenix, Inc. (Fremont, Calif.),HuMAb-Mouse® and TC Mouse™ from Medarex, Inc. (Princeton, N.J.), and theVelocImmune® mouse from Regeneron Pharmaceuticals, Inc. (Tarrytown,N.Y.).

In an alternative, antibodies may be made recombinantly and expressedusing any method known in the art. In another alternative, antibodiesmay be made recombinantly by phage display technology. See, for example,U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; andWinter et al., 1994, Annu. Rev. Immunol. 12:433-455. Alternatively, thephage display technology (McCafferty et al., 1990, Nature 348:552-553)can be used to produce human antibodies and antibody fragments in vitro,from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors. According to this technique, antibody V domain genesare cloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, such as M13 or fd, and displayed asfunctional antibody fragments on the surface of the phage particle.Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. Thus, the phage mimics some of theproperties of the B cell. Phage display can be performed in a variety offormats; see, e.g., Johnson, Kevin S, and Chiswell, David J., 1993,Current Opinion in Structural Biology 3:564-571. Several sources ofV-gene segments can be used for phage display. Clackson et al., 1991,Nature 352:624-628 isolated a diverse array of anti-oxazolone antibodiesfrom a small random combinatorial library of V genes derived from thespleens of immunized mice. A repertoire of V genes from unimmunizedhuman donors can be constructed and antibodies to a diverse array ofantigens (including self-antigens) can be isolated essentially followingthe techniques described by Mark et al., 1991, J. Mol. Biol.222:581-597, or Griffith et al., 1993, EMBO J. 12:725-734. In a naturalimmune response, antibody genes accumulate mutations at a high rate(somatic hypermutation). Some of the changes introduced will conferhigher affinity, and B cells displaying high-affinity surfaceimmunoglobulin are preferentially replicated and differentiated duringsubsequent antigen challenge. This natural process can be mimicked byemploying the technique known as “chain shuffling.” (Marks et al., 1992,Bio/Technol. 10:779-783). In this method, the affinity of “primary”human antibodies obtained by phage display can be improved bysequentially replacing the heavy and light chain V region genes withrepertoires of naturally occurring variants (repertoires) of V domaingenes obtained from unimmunized donors. This technique allows theproduction of antibodies and antibody fragments with affinities in thepM-nM range. A strategy for making very large phage antibody repertoires(also known as “the mother-of-all libraries”) has been described byWaterhouse et al., 1993, Nucl. Acids Res. 21:2265-2266. Gene shufflingcan also be used to derive human antibodies from rodent antibodies,where the human antibody has similar affinities and specificities to thestarting rodent antibody. According to this method, which is alsoreferred to as “epitope imprinting”, the heavy or light chain V domaingene of rodent antibodies obtained by phage display technique isreplaced with a repertoire of human V domain genes, creatingrodent-human chimeras. Selection on antigen results in isolation ofhuman variable regions capable of restoring a functional antigen-bindingsite, i.e., the epitope governs (imprints) the choice of partner. Whenthe process is repeated in order to replace the remaining rodent Vdomain, a human antibody is obtained (see PCT Publ. No. WO 93/06213).Unlike traditional humanization of rodent antibodies by CDR grafting,this technique provides completely human antibodies, which have noframework or CDR residues of rodent origin.

It is apparent that although the above discussion pertains to humanizedantibodies, the general principles discussed are applicable tocustomizing antibodies for use, for example, in dogs, cats, primate,equines and bovines. It is further apparent that one or more aspects ofhumanizing an antibody described herein may be combined, e.g., CDRgrafting, framework mutation and CDR mutation.

Antibodies may be made recombinantly by first isolating the antibodiesand antibody producing cells from host animals, obtaining the genesequence, and using the gene sequence to express the antibodyrecombinantly in host cells (e.g., CHO cells). Another method which maybe employed is to express the antibody sequence in plants (e.g.,tobacco) or transgenic milk. Methods for expressing antibodiesrecombinantly in plants or milk have been disclosed. See, for example,Peeters, 2001, et al. Vaccine 19:2756; Lonberg, N. and D. Huszar, 1995,Int. Rev. Immunol 13:65; and Pollock, et al., 1999, J Immunol Methods231:147. Methods for making derivatives of antibodies, e.g., humanized,single chain, etc. are known in the art.

Immunoassays and flow cytometry sorting techniques such as fluorescenceactivated cell sorting (FACS) can also be employed to isolate antibodiesthat are specific for PCSK9.

The antibodies can be bound to many different carriers. Carriers can beactive and/or inert. Examples of well-known carriers includepolypropylene, polystyrene, polyethylene, dextran, nylon, amylases,glass, natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation. In some embodiments, thecarrier comprises a moiety that targets the myocardium.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors (such as expression vectors disclosed in PCTPubl. No. WO 87/04462), which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. See, e.g., PCT Publ. No. WO 87/04462. The DNA also may bemodified, for example, by substituting the coding sequence for humanheavy and light chain constant domains in place of the homologous murinesequences, Morrison et al., 1984, Proc. Nat. Acad. Sci. 81:6851, or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. In thatmanner, “chimeric” or “hybrid” antibodies are prepared that have thebinding specificity of a PCSK9 monoclonal antibody herein.

PCSK9 antagonist antibodies and polypeptides derived from antibodies canbe identified or characterized using methods known in the art, wherebyreduction, amelioration, or neutralization of PCSK9 biological activityis detected and/or measured. In some embodiments, a PCSK9 antagonistantibody or polypeptide is identified by incubating a candidate agentwith PCSK9 and monitoring binding and/or attendant reduction orneutralization of a biological activity of PCSK9. The binding assay maybe performed with purified PCSK9 polypeptide(s), or with cells naturallyexpressing, or transfected to express, PCSK9 polypeptide(s). In oneembodiment, the binding assay is a competitive binding assay, where theability of a candidate antibody to compete with a known PCSK9 antagonistfor PCSK9 binding is evaluated. The assay may be performed in variousformats, including the ELISA format. In other embodiments, a PCSK9antagonist antibody is identified by incubating a candidate agent withPCSK9 and monitoring binding and attendant inhibition of LDLR expressionand/or blood cholesterol clearance.

Following initial identification, the activity of a candidate PCSK9antagonist antibody can be further confirmed and refined by bioassaysthat are known to test the targeted biological activities.Alternatively, bioassays can be used to screen candidates directly. Someof the methods for identifying and characterizing PCSK9 antagonistantibodies, peptides, or aptamers are described in detail in theExamples.

PCSK9 antagonist antibodies may be characterized using methods wellknown in the art. For example, one method is to identify the epitope towhich it binds, or “epitope mapping.” There are many methods known inthe art for mapping and characterizing the location of epitopes onproteins, including solving the crystal structure of an antibody-antigencomplex, competition assays, gene fragment expression assays, andsynthetic peptide-based assays, as described, for example, in Chapter 11of Harlow and Lane, Using Antibodies, a Laboratory Manual, (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1999). In anadditional example, epitope mapping can be used to determine thesequence to which a PCSK9 antagonist antibody binds. Epitope mapping iscommercially available from various sources, for example, PepscanSystems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitopecan be a linear epitope, i.e., contained in a single stretch of aminoacids, or a conformational epitope formed by a three-dimensionalinteraction of amino acids that may not necessarily be contained in asingle stretch. Peptides of varying lengths (e.g., at least 4-6 aminoacids long) can be isolated or synthesized (e.g., recombinantly) andused for binding assays with a PCSK9 antagonist antibody. In anotherexample, the epitope to which the PCSK9 antagonist antibody binds can bedetermined in a systematic screening by using overlapping peptidesderived from the PCSK9 sequence and determining binding by the PCSK9antagonist antibody. According to the gene fragment expression assays,the open reading frame encoding PCSK9 is fragmented either randomly orby specific genetic constructions and the reactivity of the expressedfragments of PCSK9 with the antibody to be tested is determined. Thegene fragments may, for example, be produced by PCR and then transcribedand translated into protein in vitro, in the presence of radioactiveamino acids. The binding of the antibody to the radioactively labeledPCSK9 fragments is then determined by immunoprecipitation and gelelectrophoresis. Certain epitopes can also be identified by using largelibraries of random peptide sequences displayed on the surface of phageparticles (phage libraries). Alternatively, a defined library ofoverlapping peptide fragments can be tested for binding to the testantibody in simple binding assays. In an additional example, mutagenesisof an antigen binding domain, domain swapping experiments and alaninescanning mutagenesis can be performed to identify residues required,sufficient, and/or necessary for epitope binding. For example, domainswapping experiments can be performed using a mutant PCSK9 in whichvarious fragments of the PCSK9 polypeptide have been replaced (swapped)with sequences from PCSK9 from another species, or a closely related,but antigenically distinct protein (such as another member of theproprotein convertase family). By assessing binding of the antibody tothe mutant PCSK9, the importance of the particular PCSK9 fragment toantibody binding can be assessed.

Yet another method which can be used to characterize a PCSK9 antagonistantibody is to use competition assays with other antibodies known tobind to the same antigen, i.e., various fragments on PCSK9, to determineif the PCSK9 antagonist antibody binds to the same epitope as otherantibodies. Competition assays are well known to those of skill in theart.

The crystal structure of the antibody and antibody:antigen complex canalso be used to characterize the antibody. The residues are identifiedby calculating the difference in accessible surface area between theL1L3:PCSK9 crystal structure and PCSK9 structure alone. PCSK9 residuesthat show buried surface area upon complex formation with L1L3 antibodyare included as a part of the epitope. The solvent accessible surface ofa protein is defined as the locus of the centre of a probe sphere(representing a solvent molecule of 1.4 Å radius) as it rolls over theVan der Waals surface of the protein. The solvent accessible surfacearea is calculated by generating surface points on an extended sphereabout each atom (at a distance from the atom centre equal to the sum ofthe atom and probe radii), and eliminating those that lie withinequivalent spheres associated with neighboring atoms as implemented inprogram AREAIMOL (Briggs, P.J., 2000, CCP4 Newsletter No. 38, CCLRC,Daresbury).

An expression vector can be used to direct expression of a PCSK9antagonist antibody. One skilled in the art is familiar withadministration of expression vectors to obtain expression of anexogenous protein in vivo. See, e.g., U.S. Pat. Nos. 6,436,908;6,413,942; and 6,376,471. Administration of expression vectors includeslocal or systemic administration, including injection, oraladministration, particle gun or catheterized administration, and topicaladministration. In another embodiment, the expression vector isadministered directly to the sympathetic trunk or ganglion, or into acoronary artery, atrium, ventrical, or pericardium.

Targeted delivery of therapeutic compositions containing an expressionvector, or subgenomic polynucleotides can also be used.Receptor-mediated DNA delivery techniques are described in, for example,Findeis et al., 1993, Trends Biotechnol. 11:202; Chiou et al., 1994,Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J.A. Wolff, ed.); Wu et al., 1988, J. Biol. Chem. 263:621; Wu et al.,1994, J. Biol. Chem. 269:542; Zenke et al., 1990, Proc. Natl. Acad. Sci.USA 87:3655; Wu et al., 1991, J. Biol. Chem. 266:338. Therapeuticcompositions containing a polynucleotide are administered in a range ofabout 100 ng to about 200 mg of DNA for local administration in a genetherapy protocol. Concentration ranges of about 500 ng to about 50 mg,about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg toabout 100 μg of DNA can also be used during a gene therapy protocol. Thetherapeutic polynucleotides and polypeptides can be delivered using genedelivery vehicles. The gene delivery vehicle can be of viral ornon-viral origin (see generally, Jolly, 1994, Cancer Gene Therapy 1:51;Kimura, 1994, Human Gene Therapy 5:845; Connelly, 1995, Human GeneTherapy 1:185; and Kaplitt, 1994, Nature Genetics 6:148). Expression ofsuch coding sequences can be induced using endogenous mammalian orheterologous promoters. Expression of the coding sequence can be eitherconstitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publ. Nos. WO 90/07936; WO 94/03622; WO93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat.Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EP Patent No.0 345 242), alphavirus-based vectors (e.g., Sindbis virus vectors,Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCCVR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCCVR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associatedvirus (AAV) vectors (see, e.g., PCT Publ. Nos. WO 94/12649, WO 93/03769;WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administrationof DNA linked to killed adenovirus as described in Curiel, 1992, Hum.Gene Ther. 3:147, can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, 1992, Hum. Gene Ther.3:147); ligand-linked DNA (see, e.g., Wu, J., 1989, Biol. Chem.264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publ. Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publ. No. WO 90/11092 and U.S.Pat. No. 5,580,859. Liposomes that can act as gene delivery vehicles aredescribed in U.S. Pat. No. 5,422,120; PCT Publ. Nos. WO 95/13796; WO94/23697; WO 91/14445; and EP 0524968. Additional approaches aredescribed in Philip, 1994, Mol. Cell. Biol., 14:2411, and in Woffendin,1994 Proc. Natl. Acad. Sci. 91:1581.

This invention encompasses compositions, including pharmaceuticalcompositions, comprising antibodies described herein or made by themethods and having the characteristics described herein. As used herein,compositions comprise one or more antibodies, peptides, or aptamers thatantagonize the interaction of PCSK9 with the LDLR, and/or one or morepolynucleotides comprising sequences encoding one or more theseantibodies or peptides. These compositions may further comprise suitableexcipients, such as pharmaceutically acceptable excipients includingbuffers, which are well known in the art.

The PCSK9 antagonist antibodies and peptides of the invention arecharacterized by any (one or more) of the following characteristics: (a)bind to PCSK9; (b) block PCSK9 interaction with the LDLR; (c) decreasePCSK9-mediated down-regulation of the LDLR; and (d) inhibitPCSK9-mediated inhibition of LDL blood clearance. Preferably, PCSK9antibodies have two or more of these features. More preferably, theantibodies have three or more of the features. Most preferably, theantibodies have all four characteristics.

Accordingly, the invention provides any of the following, orcompositions (including pharmaceutical compositions) comprising anyantibody having a partial light chain sequence and a partial heavy chainsequence as found in Table 1. The underlined sequences are CDR sequencesaccording to Kabat and in bold according to Chothia.

TABLE 1 Light Chain Heavy Chain mAb Variable Region Variable Region 4A5DIVMTQSQKFMSTSVGDRV EVQLQQSGPELVKPGASVKISCKAS SVTC KASQNVGTNVA WYQ GYTFTDYYMNWVKQSHGKSLEWIG QKPGQSPKALIY SASYRYS G DINPNNGGTTYNQKFKGKATLTVDKSVPDRFTGSGSGTDFTLTISN YSTAYMELRSLTSEDSAVYYCAR WL VLSEDLAEYFC QQFYSYPYTLFAY WGQGTLVTVSA FGGGTKLEIK (SEQ ID NO: 20) (SEQ ID NO: 16) 5A10DIVMTQSHKFMSTSVGDRVS QVQLQQPGAELVKPGASVKLSCKAS ITC KASQDVSTAVA WYQQKGYTFT SYWMHWVKQRPGQGLEWIG PGQSPKLLIY SASYRYT GVPEINPSNGRTNYNEKFKSKATLTVDKS DRFTGSGSGTDFTFTISSVQ SSTAYMQLSSLTSEDSAVYYCARER AEDLAVYYC QQRYSTPRT F PLYAMDY WGQGTSVTVSS GGGTKLEIK (SEQ ID NO: 21)(SEQ ID NO: 17) 6F6 DIQMTQTTSSLSASLGDRVTI EVQLQQSGPELVKPGASVKISCKAS SCSASQGISNYLN WYQQKP GYTFT DYYMNWVKQSHGKSLEWIG DGTVKLLIY YTSSLHS GVPSDINPNNGGTSYNQKFKGKATLTVDK RFSGSGSGTDYSLTISNLEP SSSTAYMELRSLTSEDSAVYYCAGG EDIATYYC QQYSKLPFT FGS GIYYRYDRNYFDY WGQGTTLTVSS GTKLEIK(SEQ ID NO: 22) (SEQ ID NO: 18) 7D4 DIVMTQSHKFMSTSFGDRVSEVKLVESEGGLVQPGSSMKLSCTAS ITC KASQDVSNALA WYQQK GFTFSDYYMAWVRQVPEKGLEWVA PGHSPKLLIF SASYRYT GVP NINYDGSNTSYLDSLKSRFIISRDNAKDRFTGSGSGTDFTFTISSVQ NILYLQMSSLKSEDTATYYCAR EKFA AEDLAVYYC QQHYSTPWT FAMDY WGQGTSVTVSS GGGTKLEIK (SEQ ID NO: 23) (SEQ ID NO: 19) L1L3DIQMTQSPSSLSASVGDRVT QVQLVQSGAEVKKPGASVKVSCKAS ITC RASQGISSALA WYQQKPGYTFT SYYMHWVRQAPGQGLEWM GKAPKLLIY SASYRYT GVPSGEISPFGGRTNYNEKFKSRVTMTRD RFSGSGSGTDFTFTISSLQP TSTSTVYMELSSLRSEDTAVYYCARE EDIATYYC QQRYSLWRT FG RPLYASDL WGQGTTVTVSS QGTKLEIK (SEQ ID NO:54)(SEQ ID NO: 53)

The invention also provides CDR portions of antibodies to PCSK9(including Chothia and Kabat CDRs). Determination of CDR regions is wellwithin the skill of the art. It is understood that in some embodiments,CDRs can be a combination of the Kabat and Chothia CDR (also termed“combined CDRs” or “extended CDRs”). In some embodiments, the CDRs arethe Kabat CDRs. In other embodiments, the CDRs are the Chothia CDRs. Inother words, in embodiments with more than one CDR, the CDRs may be anyof Kabat, Chothia, combination CDRs, or combinations thereof.

The invention also provides methods of making any of these antibodies orpolypeptides. The antibodies of this invention can be made by proceduresknown in the art. The polypeptides can be produced by proteolytic orother degradation of the antibodies, by recombinant methods (i.e.,single or fusion polypeptides) as described above or by chemicalsynthesis. Polypeptides of the antibodies, especially shorterpolypeptides up to about 50 amino acids, are conveniently made bychemical synthesis. Methods of chemical synthesis are known in the artand are commercially available. For example, an antibody could beproduced by an automated polypeptide synthesizer employing the solidphase method. See also, U.S. Pat. Nos. 5,807,715; 4,816,567; and6,331,415.

In another alternative, the antibodies and peptides can be maderecombinantly using procedures that are well known in the art. In oneembodiment, a polynucleotide comprises a sequence encoding the heavychain and/or the light chain variable regions of antibody 4A5, 5A10,6F6, 7D4 or L1L3. The sequence encoding the antibody of interest may bemaintained in a vector in a host cell and the host cell can then beexpanded and frozen for future use. Vectors (including expressionvectors) and host cells are further described herein.

The invention also encompasses scFv of antibodies of this invention.Single chain variable region fragments are made by linking light and/orheavy chain variable regions by using a short linking peptide. Bird etal., 1988, Science 242:423-426. An example of a linking peptide is(GGGGS)₃ (SEQ ID NO:24), which bridges approximately 3.5 nm between thecarboxy terminus of one variable region and the amino terminus of theother variable region. Linkers of other sequences have been designed andused. Bird et al., 1988, supra. Linkers should be short, flexiblepolypeptides and preferably comprised of less than about 20 amino acidresidues. Linkers can in turn be modified for additional functions, suchas attachment of drugs or attachment to solid supports. The single chainvariants can be produced either recombinantly or synthetically. Forsynthetic production of scFv, an automated synthesizer can be used. Forrecombinant production of scFv, a suitable plasmid containingpolynucleotide that encodes the scFv can be introduced into a suitablehost cell, either eukaryotic, such as yeast, plant, insect or mammaliancells, or prokaryotic, such as E. coli. Polynucleotides encoding thescFv of interest can be made by routine manipulations such as ligationof polynucleotides. The resultant scFv can be isolated using standardprotein purification techniques known in the art.

Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al., 1993, Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al., 1994, Structure 2:1121-1123).

For example, bispecific antibodies, monoclonal antibodies that havebinding specificities for at least two different antigens, can beprepared using the antibodies disclosed herein. Methods for makingbispecific antibodies are known in the art (see, e.g., Suresh et al.,1986, Methods in Enzymology 121:210). Traditionally, the recombinantproduction of bispecific antibodies was based on the coexpression of twoimmunoglobulin heavy chain-light chain pairs, with the two heavy chainshaving different specificities (Millstein and Cuello, 1983, Nature 305,537-539).

According to one approach to making bispecific antibodies, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantdomain sequences. The fusion preferably is with an immunoglobulin heavychain constant domain, comprising at least part of the hinge, CH2 andCH3 regions. It is preferred to have the first heavy chain constantregion (CH1), containing the site necessary for light chain binding,present in at least one of the fusions. DNAs encoding the immunoglobulinheavy chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are cotransfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In one approach, the bispecific antibodies are composed of a hybridimmunoglobulin heavy chain with a first binding specificity in one arm,and a hybrid immunoglobulin heavy chain-light chain pair (providing asecond binding specificity) in the other arm. This asymmetric structure,with an immunoglobulin light chain in only one half of the bispecificmolecule, facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations. This approach isdescribed in PCT Publ. No. WO 94/04690.

Heteroconjugate antibodies, comprising two covalently joined antibodies,are also within the scope of the invention. Such antibodies have beenused to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (PCT Publ. Nos. WO91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents and techniques are well known in the art, and are described inU.S. Pat. No. 4,676,980.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods of synthetic protein chemistry, including those involvingcross-linking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

Humanized antibody comprising one or more CDRs of antibodies 5A10 or 7D4or one or more CDRs derived from antibodies 5A10 or 7D4 can be made, forexample, using any methods know in the art. For example, four generalsteps may be used to humanize a monoclonal antibody. These are: (1)determining the nucleotide and predicted amino acid sequence of thestarting antibody light and heavy variable domains; (2) designing thehumanized antibody, i.e., deciding which antibody framework region touse during the humanizing process; (3) using the actual humanizingmethodologies/techniques; and (4) transfecting and expressing thehumanized antibody. See, for example, U.S. Pat. Nos. 4,816,567;5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762;5,585,089; and 6,180,370.

In the recombinant humanized antibodies, the Fc portion can be modifiedto avoid interaction with Fcγ receptor and the complement and immunesystems. The techniques for preparation of such antibodies are describedin WO 99/58572. For example, the constant region may be engineered tomore resemble human constant regions to avoid immune response if theantibody is used in clinical trials and treatments in humans. See, forexample, U.S. Pat. Nos. 5,997,867 and 5,866,692.

Humanized antibody comprising the light or heavy chain variable regionsor one or more CDRs of an antibody or its variants shown in Table 1, orone or more CDRs derived from the antibody or its variants shown inTable 2 can be made using any methods known in the art.

Humanized antibodies may be made by any method known in the art.

The invention encompasses modifications to the antibodies andpolypeptides of the invention variants shown in Table 1, includingfunctionally equivalent antibodies which do not significantly affecttheir properties and variants which have enhanced or decreased activityand/or affinity. For example, the amino acid sequence may be mutated toobtain an antibody with the desired binding affinity to PCSK9.Modification of polypeptides is routine practice in the art and need notbe described in detail herein. Modification of polypeptides isexemplified in the Examples. Examples of modified polypeptides includepolypeptides with conservative substitutions of amino acid residues, oneor more deletions or additions of amino acids which do not significantlydeleteriously change the functional activity, or which mature (enhance)the affinity of the polypeptide for its ligand, or use of chemicalanalogs.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto an epitope tag. Other insertional variants of the antibody moleculeinclude the fusion to the N- or C-terminus of the antibody of an enzymeor a polypeptide which increases the half-life of the antibody in theblood circulation.

Substitution variants have at least one amino acid residue in theantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 2 under the heading of“conservative substitutions.” If such substitutions result in a changein biological activity, then more substantial changes, denominated“exemplary substitutions” in Table 2, or as further described below inreference to amino acid classes, may be introduced and the productsscreened.

TABLE 2 Amino Acid Substitutions Conservative Original ResidueSubstitutions Exemplary Substitutions Ala (A) Val Val; Leu; Ile Arg (R)Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp, Lys; Arg Asp (D) Glu Glu;Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; Glu Glu (E) Asp Asp; Gln Gly(G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val; Met;Ala; Phe; Norleucine Leu (L) Ile Norleucine; Ile; Val; Met; Ala; Phe Lys(K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val;Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W)Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met;Phe; Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

-   -   (1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) Polar without charge: Cys, Ser, Thr, Asn, Gln;    -   (3) Acidic (negatively charged): Asp, Glu;    -   (4) Basic (positively charged): Lys, Arg;    -   (5) Residues that influence chain orientation: Gly, Pro; and    -   (6) Aromatic: Trp, Tyr, Phe, His.

Non-conservative substitutions are made by exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcross-linking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability, particularly where the antibody is an antibodyfragment such as an Fv fragment.

Amino acid modifications can range from changing or modifying one ormore amino acids to complete redesign of a region, such as the variableregion. Changes in the variable region can alter binding affinity and/orspecificity. In some embodiments, no more than one to five conservativeamino acid substitutions are made within a CDR domain. In otherembodiments, no more than one to three conservative amino acidsubstitutions are made within a CDR domain. In still other embodiments,the CDR domain is CDR H3 and/or CDR L3.

Modifications also include glycosylated and nonglycosylatedpolypeptides, as well as polypeptides with other post-translationalmodifications, such as, for example, glycosylation with differentsugars, acetylation, and phosphorylation. Antibodies are glycosylated atconserved positions in their constant regions (Jefferis and Lund, 1997,Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32).The oligosaccharide side chains of the immunoglobulins affect theprotein's function (Boyd et al., 1996, Mol. Immunol. 32:1311-1318;Wittwe and Howard, 1990, Biochem. 29:4175-4180) and the intramolecularinteraction between portions of the glycoprotein, which can affect theconformation and presented three-dimensional surface of the glycoprotein(Jefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech.7:409-416). Oligosaccharides may also serve to target a givenglycoprotein to certain molecules based upon specific recognitionstructures. Glycosylation of antibodies has also been reported to affectantibody-dependent cellular cytotoxicity (ADCC). In particular, CHOcells with tetracycline-regulated expression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GlcNAc, wasreported to have improved ADCC activity (Umana et al., 1999, NatureBiotech. 17:176-180).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

The glycosylation pattern of antibodies may also be altered withoutaltering the underlying nucleotide sequence. Glycosylation largelydepends on the host cell used to express the antibody. Since the celltype used for expression of recombinant glycoproteins, e.g., antibodies,as potential therapeutics is rarely the native cell, variations in theglycosylation pattern of the antibodies can be expected (see, e.g., Hseet al., 1997, J. Biol. Chem. 272:9062-9070).

In addition to the choice of host cells, factors that affectglycosylation during recombinant production of antibodies include growthmode, media formulation, culture density, oxygenation, pH, purificationschemes and the like. Various methods have been proposed to alter theglycosylation pattern achieved in a particular host organism includingintroducing or overexpressing certain enzymes involved inoligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and5,278,299). Glycosylation, or certain types of glycosylation, can beenzymatically removed from the glycoprotein, for example, usingendoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1,endoglycosidase F2, endoglycosidase F3. In addition, the recombinanthost cell can be genetically engineered to be defective in processingcertain types of polysaccharides. These and similar techniques are wellknown in the art.

Other methods of modification include using coupling techniques known inthe art, including, but not limited to, enzymatic means, oxidativesubstitution and chelation. Modifications can be used, for example, forattachment of labels for immunoassay. Modified polypeptides are madeusing established procedures in the art and can be screened usingstandard assays known in the art, some of which are described below andin the Examples.

In some embodiments of the invention, the antibody comprises a modifiedconstant region, such as a constant region that is immunologically inertor partially inert, e.g., does not trigger complement mediated lysis,does not stimulate ADCC, or does not activate microglia; or have reducedactivities (compared to the unmodified antibody) in any one or more ofthe following: triggering complement mediated lysis, stimulating ADCC,or activating microglia. Different modifications of the constant regionmay be used to achieve optimal level and/or combination of effectorfunctions. See, for example, Morgan et al., 1995, Immunology 86:319-324;Lund et al., 1996, J. Immunology 157:4963-9 157:4963-4969; Idusogie etal., 2000, J. Immunology 164:4178-4184; Tao et al., 1989, J. Immunology143: 2595-2601; and Jefferis et al., 1998, Immunological Reviews163:59-76. In some embodiments, the constant region is modified asdescribed in Eur. J. Immunol., 1999, 29:2613-2624; PCT Publ. No.WO99/58572; and/or UK Patent Application No. 9809951.8. In otherembodiments, the antibody comprises a human heavy chain IgG2 constantregion comprising the following mutations: A330P331 to S330S331 (aminoacid numbering with reference to the wild type IgG2 sequence). Eur. J.Immunol., 1999, 29:2613-2624. In still other embodiments, the constantregion is aglycosylated for N-linked glycosylation. In some embodiments,the constant region is aglycosylated for N-linked glycosylation bymutating the glycosylated amino acid residue or flanking residues thatare part of the N-glycosylation recognition sequence in the constantregion. For example, N-glycosylation site N297 may be mutated to A, Q,K, or H. See, Tao et al., 1989, J. Immunology 143: 2595-2601; andJefferis et al., 1998, Immunological Reviews 163:59-76. In someembodiments, the constant region is aglycosylated for N-linkedglycosylation. The constant region may be aglycosylated for N-linkedglycosylation enzymatically (such as removing carbohydrate by enzymePNGase), or by expression in a glycosylation deficient host cell.

Other antibody modifications include antibodies that have been modifiedas described in PCT Publ. No. WO 99/58572. These antibodies comprise, inaddition to a binding domain directed at the target molecule, aneffector domain having an amino acid sequence substantially homologousto all or part of a constant domain of a human immunoglobulin heavychain. These antibodies are capable of binding the target moleculewithout triggering significant complement dependent lysis, orcell-mediated destruction of the target. In some embodiments, theeffector domain is capable of specifically binding FcRn and/or FcγRIIb.These are typically based on chimeric domains derived from two or morehuman immunoglobulin heavy chain C_(H)2 domains. Antibodies modified inthis manner are particularly suitable for use in chronic antibodytherapy, to avoid inflammatory and other adverse reactions toconventional antibody therapy.

The invention includes affinity matured embodiments. For example,affinity matured antibodies can be produced by procedures known in theart (Marks et al., 1992, Bio/Technology, 10:779-783; Barbas et al.,1994, Proc Nat. Acad. Sci, USA 91:3809-3813; Schier et al., 1995, Gene,169:147-155; Yelton et al., 1995, J. Immunol., 155:1994-2004; Jackson etal., 1995, J. Immunol., 154(7):3310-9; Hawkins et al., 1992, J. Mol.Biol., 226:889-896; and PCT Publ. No. WO2004/058184).

The following methods may be used for adjusting the affinity of anantibody and for characterizing a CDR. One way of characterizing a CDRof an antibody and/or altering (such as improving) the binding affinityof a polypeptide, such as an antibody, termed “library scanningmutagenesis”. Generally, library scanning mutagenesis works as follows.One or more amino acid positions in the CDR are replaced with two ormore (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20) amino acids using art recognized methods. This generatessmall libraries of clones (in some embodiments, one for every amino acidposition that is analyzed), each with a complexity of two or moremembers (if two or more amino acids are substituted at every position).Generally, the library also includes a clone comprising the native(unsubstituted) amino acid. A small number of clones, e.g., about 20-80clones (depending on the complexity of the library), from each libraryare screened for binding affinity to the target polypeptide (or otherbinding target), and candidates with increased, the same, decreased, orno binding are identified. Methods for determining binding affinity arewell-known in the art. Binding affinity may be determined using Biacoresurface plasmon resonance analysis, which detects differences in bindingaffinity of about 2-fold or greater. Biacore is particularly useful whenthe starting antibody already binds with a relatively high affinity, forexample a K_(D) of about 10 nM or lower. Screening using Biacore surfaceplasmon resonance is described in the Examples, herein.

Binding affinity may be determined using Kinexa Biocensor, scintillationproximity assays, ELISA, ORIGEN immunoassay (IGEN), fluorescencequenching, fluorescence transfer, and/or yeast display. Binding affinitymay also be screened using a suitable bioassay.

In some embodiments, every amino acid position in a CDR is replaced (insome embodiments, one at a time) with all 20 natural amino acids usingart recognized mutagenesis methods (some of which are described herein).This generates small libraries of clones (in some embodiments, one forevery amino acid position that is analyzed), each with a complexity of20 members (if all 20 amino acids are substituted at every position).

In some embodiments, the library to be screened comprises substitutionsin two or more positions, which may be in the same CDR or in two or moreCDRs. Thus, the library may comprise substitutions in two or morepositions in one CDR. The library may comprise substitution in two ormore positions in two or more CDRs. The library may comprisesubstitution in 3, 4, 5, or more positions, said positions found in two,three, four, five or six CDRs. The substitution may be prepared usinglow redundancy codons. See, e.g., Table 2 of Balint et al., 1993, Gene137(1):109-18).

The CDR may be CDRH3 and/or CDRL3. The CDR may be one or more of CDRL1,CDRL2, CDRL3, CDRH1, CDRH2, and/or CDRH3. The CDR may be a Kabat CDR, aChothia CDR, or an extended CDR.

Candidates with improved binding may be sequenced, thereby identifying aCDR substitution mutant which results in improved affinity (also termedan “improved” substitution). Candidates that bind may also be sequenced,thereby identifying a CDR substitution which retains binding.

Multiple rounds of screening may be conducted. For example, candidates(each comprising an amino acid substitution at one or more position ofone or more CDR) with improved binding are also useful for the design ofa second library containing at least the original and substituted aminoacid at each improved CDR position (i.e., amino acid position in the CDRat which a substitution mutant showed improved binding). Preparation,screening, and selection of this library is discussed further below.

Library scanning mutagenesis also provides a means for characterizing aCDR, in so far as the frequency of clones with improved binding, thesame binding, decreased binding or no binding also provide informationrelating to the importance of each amino acid position for the stabilityof the antibody-antigen complex. For example, if a position of the CDRretains binding when changed to all 20 amino acids, that position isidentified as a position that is unlikely to be required for antigenbinding. Conversely, if a position of CDR retains binding in only asmall percentage of substitutions, that position is identified as aposition that is important to CDR function. Thus, the library scanningmutagenesis methods generate information regarding positions in the CDRsthat can be changed to many different amino acids (including all 20amino acids), and positions in the CDRs which cannot be changed or whichcan only be changed to a few amino acids.

Candidates with improved affinity may be combined in a second library,which includes the improved amino acid, the original amino acid, and mayfurther include additional substitutions at that position, depending onthe complexity of the library that is desired, or permitted using thedesired screening or selection method. In addition, if desired, andadjacent amino acid position can be randomized to at least two or moreamino acids. Randomization of adjacent amino acids may permit additionalconformational flexibility in the mutant CDR, which may, in turn, permitor facilitate the introduction of a larger number of improvingmutations. The library may also comprise substitution at positions thatdid not show improved affinity in the first round of screening.

The second library is screened or selected for library members withimproved and/or altered binding affinity using any method known in theart, including screening using Biacore surface plasmon resonanceanalysis, and selection using any method known in the art for selection,including phage display, yeast display, and ribosome display.

The invention also encompasses fusion proteins comprising one or morefragments or regions from the antibodies or polypeptides of thisinvention. In one embodiment, a fusion polypeptide is provided thatcomprises at least 10 contiguous amino acids of a variable light chainregion shown in SEQ ID NOs: 53, 16, 17, 18, or 19 and/or at least 10amino acids of a variable heavy chain region shown in SEQ ID NOs: 54,20, 21, 22, or 23. In other embodiments, a fusion polypeptide isprovided that comprises at least about 10, at least about 15, at leastabout 20, at least about 25, or at least about 30 contiguous amino acidsof the variable light chain region and/or at least about 10, at leastabout 15, at least about 20, at least about 25, or at least about 30contiguous amino acids of the variable heavy chain region. In anotherembodiment, the fusion polypeptide comprises a light chain variableregion and/or a heavy chain variable region, as shown in any of thesequence pairs selected from among SEQ ID NOs: 53 and 54, 16 and 20, 17and 21, 18 and 22, and 19 and 23. In another embodiment, the fusionpolypeptide comprises one or more CDR(s). In still other embodiments,the fusion polypeptide comprises CDR H3 (VH CDR3) and/or CDR L3 (VLCDR3). For purposes of this invention, a fusion protein contains one ormore antibodies and another amino acid sequence to which it is notattached in the native molecule, for example, a heterologous sequence ora homologous sequence from another region. Exemplary heterologoussequences include, but are not limited to a “tag” such as a FLAG tag ora 6H is tag. Tags are well known in the art.

A fusion polypeptide can be created by methods known in the art, forexample, synthetically or recombinantly. Typically, the fusion proteinsof this invention are made by preparing an expressing a polynucleotideencoding them using recombinant methods described herein, although theymay also be prepared by other means known in the art, including, forexample, chemical synthesis.

This invention also provides compositions comprising antibodies orpolypeptides conjugated (for example, linked) to an agent thatfacilitate coupling to a solid support (such as biotin or avidin). Forsimplicity, reference will be made generally to antibodies with theunderstanding that these methods apply to any of the PCSK9 bindingand/or antagonist embodiments described herein. Conjugation generallyrefers to linking these components as described herein. The linking(which is generally fixing these components in proximate association atleast for administration) can be achieved in any number of ways. Forexample, a direct reaction between an agent and an antibody is possiblewhen each possesses a substituent capable of reacting with the other.For example, a nucleophilic group, such as an amino or sulfhydryl group,on one may be capable of reacting with a carbonyl-containing group, suchas an anhydride or an acid halide, or with an alkyl group containing agood leaving group (e.g., a halide) on the other.

An antibody or polypeptide of this invention may be linked to a labelingagent such as a fluorescent molecule, a radioactive molecule or anyothers labels known in the art. Labels are known in the art whichgenerally provide (either directly or indirectly) a signal.

The invention also provides compositions (including pharmaceuticalcompositions) and kits comprising, as this disclosure makes clear, anyor all of the antibodies and/or polypeptides described herein.

The invention also provides isolated polynucleotides encoding theantibodies and peptides of the invention, and vectors and host cellscomprising the polynucleotide.

Accordingly, the invention provides polynucleotides (or compositions,including pharmaceutical compositions), comprising polynucleotidesencoding any of the following: the antibodies 4A5, 5A10, 6F6, 7D4, L1L3,or any fragment or part thereof having the ability to antagonize PCSK9.

In another aspect, the invention provides polynucleotides encoding anyof the antibodies (including antibody fragments) and polypeptidesdescribed herein, such as antibodies and polypeptides having impairedeffector function. Polynucleotides can be made and expressed byprocedures known in the art.

In another aspect, the invention provides compositions (such aspharmaceutical compositions) comprising any of the polynucleotides ofthe invention. In some embodiments, the composition comprises anexpression vector comprising a polynucleotide encoding the antibody asdescribed herein. In other embodiment, the composition comprises anexpression vector comprising a polynucleotide encoding any of theantibodies or polypeptides described herein. In still other embodiments,the composition comprises either or both of the polynucleotides shown inSEQ ID NO:25 and SEQ ID NO:26. Expression vectors, and administration ofpolynucleotide compositions are further described herein.

In another aspect, the invention provides a method of making any of thepolynucleotides described herein.

Polynucleotides complementary to any such sequences are also encompassedby the present invention. Polynucleotides may be single-stranded (codingor antisense) or double-stranded, and may be DNA (genomic, cDNA orsynthetic) or RNA molecules. RNA molecules include HnRNA molecules,which contain introns and correspond to a DNA molecule in a one-to-onemanner, and mRNA molecules, which do not contain introns. Additionalcoding or non-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an antibody or a portion thereof) or may comprisea variant of such a sequence. Polynucleotide variants contain one ormore substitutions, additions, deletions and/or insertions such that theimmunoreactivity of the encoded polypeptide is not diminished, relativeto a native immunoreactive molecule. The effect on the immunoreactivityof the encoded polypeptide may generally be assessed as describedherein. Variants preferably exhibit at least about 70% identity, morepreferably, at least about 80% identity, yet more preferably, at leastabout 90% identity, and most preferably, at least about 95% identity toa polynucleotide sequence that encodes a native antibody or a portionthereof.

Two polynucleotide or polypeptide sequences are said to be “identical”if the sequence of nucleotides or amino acids in the two sequences isthe same when aligned for maximum correspondence as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, or 40 to about 50, in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O., 1978, A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure (National BiomedicalResearch Foundation, Washington D.C.), Vol. 5, Suppl. 3, pp. 345-358;Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, (Academic Press, Inc., San Diego,Calif.); Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers,E. W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb.Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425;Sneath, P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy thePrinciples and Practice of Numerical Taxonomy (Freeman Press, SanFrancisco, Calif.); Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl.Acad. Sci. USA 80:726-730.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e. the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Variants may also, or alternatively, be substantially homologous to anative gene, or a portion or complement thereof. Such polynucleotidevariants are capable of hybridizing under moderately stringentconditions to a naturally occurring DNA sequence encoding a nativeantibody (or a complementary sequence).

Suitable “moderately stringent conditions” include prewashing in asolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS.

As used herein, “highly stringent conditions” or “high stringencyconditions” are those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present invention.Further, alleles of the genes comprising the polynucleotide sequencesprovided herein are within the scope of the present invention. Allelesare endogenous genes that are altered as a result of one or moremutations, such as deletions, additions and/or substitutions ofnucleotides. The resulting mRNA and protein may, but need not, have analtered structure or function. Alleles may be identified using standardtechniques (such as hybridization, amplification and/or databasesequence comparison).

The polynucleotides of this invention can be obtained using chemicalsynthesis, recombinant methods, or PCR. Methods of chemicalpolynucleotide synthesis are well known in the art and need not bedescribed in detail herein. One of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to produce adesired DNA sequence.

For preparing polynucleotides using recombinant methods, apolynucleotide comprising a desired sequence can be inserted into asuitable vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification, as furtherdiscussed herein. Polynucleotides may be inserted into host cells by anymeans known in the art. Cells are transformed by introducing anexogenous polynucleotide by direct uptake, endocytosis, transfection,F-mating or electroporation. Once introduced, the exogenouspolynucleotide can be maintained within the cell as a non-integratedvector (such as a plasmid) or integrated into the host cell genome. Thepolynucleotide so amplified can be isolated from the host cell bymethods well known within the art. See, e.g., Sambrook et al., 1989,supra.

Alternatively, PCR allows reproduction of DNA sequences. PCR technologyis well known in the art and is described in U.S. Pat. Nos. 4,683,195,4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase ChainReaction, Mullis et al., 1994, eds. (Birkauswer Press, Boston, Mass.).

RNA can be obtained by using the isolated DNA in an appropriate vectorand inserting it into a suitable host cell. When the cell replicates andthe DNA is transcribed into RNA, the RNA can then be isolated usingmethods well known to those of skill in the art, as set forth inSambrook et al., 1989, supra, for example.

Suitable cloning vectors may be constructed according to standardtechniques, or may be selected from a large number of cloning vectorsavailable in the art. While the cloning vector selected may varyaccording to the host cell intended to be used, useful cloning vectorswill generally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, and/or may carry genesfor a marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pUC18,pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp 18, mp 19,pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such aspSA3 and pAT28. These and many other cloning vectors are available fromcommercial vendors such as BioRad, Strategene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructsthat contain a polynucleotide according to the invention. It is impliedthat an expression vector must be replicable in the host cells either asepisomes or as an integral part of the chromosomal DNA. Suitableexpression vectors include but are not limited to plasmids, viralvectors, including adenoviruses, adeno-associated viruses, retroviruses,cosmids, and expression vector(s) disclosed in PCT Publ. No. WO87/04462. Vector components may generally include, but are not limitedto, one or more of the following: a signal sequence; an origin ofreplication; one or more marker genes; suitable transcriptionalcontrolling elements (such as promoters, enhancers and terminator). Forexpression (i.e., translation), one or more translational controllingelements are also usually required, such as ribosome binding sites,translation initiation sites, and stop codons.

The vectors containing the polynucleotides of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell.

The invention also provides host cells comprising any of thepolynucleotides described herein. Any host cells capable ofover-expressing heterologous DNAs can be used for the purpose ofisolating the genes encoding the antibody, polypeptide or protein ofinterest. Non-limiting examples of mammalian host cells include but arenot limited to COS, HeLa, NSO, and CHO cells. See also PCT Publ. No. WO87/04462. Suitable non-mammalian host cells include prokaryotes (such asE. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; orK. lactis). Preferably, the host cells express the cDNAs at a level ofabout 5 fold higher, more preferably, 10 fold higher, even morepreferably, 20 fold higher than that of the corresponding endogenousantibody or protein of interest, if present, in the host cells.Screening the host cells for a specific binding to PCSK9 or a PCSK9domain is effected by an immunoassay or FACS. A cell overexpressing theantibody or protein of interest can be identified.

C. Compositions

The compositions used in the methods of the invention comprise aneffective amount of a PCSK9 antagonist antibody, a PCSK9 antagonistantibody derived polypeptide, or other PCSK9 antagonists describedherein. Examples of such compositions, as well as how to formulate them,are also described in an earlier section and below. In one embodiment,the composition further comprises a PCSK9 antagonist. In anotherembodiment, the composition comprises one or more PCSK9 antagonistantibodies. In other embodiments, the PCSK9 antagonist antibodyrecognizes human PCSK9. In still other embodiments, the PCSK9 antagonistantibody is humanized. In yet other embodiments, the PCSK9 antagonistantibody comprises a constant region that does not trigger an unwantedor undesirable immune response, such as antibody-mediated lysis or ADCC.In other embodiments, the PCSK9 antagonist antibody comprises one ormore CDR(s) of the antibody (such as one, two, three, four, five, or, insome embodiments, all six CDRs). In some embodiments, the PCSK9antagonist antibody is human.

It is understood that the compositions can comprise more than one PCSK9antagonist antibody (e.g., a mixture of PCSK9 antagonist antibodies thatrecognize different epitopes of PCSK9). Other exemplary compositionscomprise more than one PCSK9 antagonist antibodies that recognize thesame epitope(s), or different species of PCSK9 antagonist antibodiesthat bind to different epitopes of PCSK9.

The composition used in the present invention can further comprisepharmaceutically acceptable carriers, excipients, or stabilizers(Remington: The Science and Practice of Pharmacy 20th Ed., 2000,Lippincott Williams and Wilkins, Ed. K. E. Hoover), in the form oflyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations, and may comprise buffers such as phosphate, citrate, andother organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrans; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Pharmaceutically acceptable excipients arefurther described herein.

In one embodiment, the antibody is administered in a formulation as asterile aqueous solution having a pH that ranges from about 5.0 to about6.5 and comprising from about 1 mg/ml to about 200 mg/ml of antibody,from about 1 millimolar to about 100 millimolar of histidine buffer,from about 0.01 mg/ml to about 10 mg/ml of polysorbate 80, from about100 millimolar to about 400 millimolar of trehalose, and from about 0.01millimolar to about 1.0 millimolar of disodium EDTA dihydrate.

The PCSK9 antagonist antibody and compositions thereof can also be usedin conjunction with other agents that serve to enhance and/or complementthe effectiveness of the agents.

D. Kits

The invention also provides kits for use in the instant methods. Kits ofthe invention include one or more containers comprising a PCSK9antagonist antibody (such as a humanized antibody) or peptide describedherein and instructions for use in accordance with any of the methods ofthe invention described herein. Generally, these instructions comprise adescription of administration of the PCSK9 antagonist antibody, peptide,or aptamer for the above described therapeutic treatments.

In some embodiments, the antibody is a humanized antibody. In someembodiments, the antibody is human. In other embodiments, the antibodyis a monoclonal antibody. The instructions relating to the use of aPCSK9 antagonist antibody generally include information as to dosage,dosing schedule, and route of administration for the intended treatment.The containers may be unit doses, bulk packages (e.g., multi-dosepackages) or sub-unit doses. Instructions supplied in the kits of theinvention are typically written instructions on a label or packageinsert (e.g., a paper sheet included in the kit), but machine-readableinstructions (e.g., instructions carried on a magnetic or opticalstorage disk) are also acceptable.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a PCSK9 antagonist antibody. The container (e.g.,pre-filled syringe or autoinjector) may further comprise a secondpharmaceutically active agent.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container.

Mutations and Modifications

To express the PCSK9 antibodies of the present invention, DNA fragmentsencoding V_(H) and V_(L) regions can first be obtained using any of themethods described above. Various modifications, e.g., mutations,deletions, and/or additions can also be introduced into the DNAsequences using standard methods known to those of skill in the art. Forexample, mutagenesis can be carried out using standard methods, such asPCR-mediated mutagenesis, in which the mutated nucleotides areincorporated into the PCR primers such that the PCR product contains thedesired mutations or site-directed mutagenesis.

One type of substitution, for example, that may be made is to change oneor more cysteines in the antibody, which may be chemically reactive, toanother residue, such as, without limitation, alanine or serine. Forexample, there can be a substitution of a non-canonical cysteine. Thesubstitution can be made in a CDR or framework region of a variabledomain or in the constant domain of an antibody. In some embodiments,the cysteine is canonical.

The antibodies may also be modified, e.g., in the variable domains ofthe heavy and/or light chains, e.g., to alter a binding property of theantibody. For example, a mutation may be made in one or more of the CDRregions to increase or decrease the K_(D) of the antibody for PCSK9, toincrease or decrease k_(off), or to alter the binding specificity of theantibody. Techniques in site-directed mutagenesis are well-known in theart. See, e.g., Sambrook et al. and Ausubel et al., supra.

A modification or mutation may also be made in a framework region orconstant domain to increase the half-life of a PCSK9 antibody. See,e.g., PCT Publ. No. WO 00/09560. A mutation in a framework region orconstant domain can also be made to alter the immunogenicity of theantibody, to provide a site for covalent or non-covalent binding toanother molecule, or to alter such properties as complement fixation,FcR binding and antibody-dependent cell-mediated cytotoxicity. Accordingto the invention, a single antibody may have mutations in any one ormore of the CDRs or framework regions of the variable domain or in theconstant domain.

In a process known as “germlining”, certain amino acids in the V_(H) andV_(L) sequences can be mutated to match those found naturally ingermline V_(H) and V_(L) sequences. In particular, the amino acidsequences of the framework regions in the V_(H) and V_(L) sequences canbe mutated to match the germline sequences to reduce the risk ofimmunogenicity when the antibody is administered. Germline DNA sequencesfor human V_(H) and V_(L) genes are known in the art (see e.g., the“Vbase” human germline sequence database; see also Kabat, E. A., et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publ. No. 91-3242;Tomlinson et al., 1992, J. Mol. Biol. 227:776-798; and Cox et al., 1994,Eur. J. Immunol. 24:827-836.

Another type of amino acid substitution that may be made is to removepotential proteolytic sites in the antibody. Such sites may occur in aCDR or framework region of a variable domain or in the constant domainof an antibody. Substitution of cysteine residues and removal ofproteolytic sites may decrease the risk of heterogeneity in the antibodyproduct and thus increase its homogeneity. Another type of amino acidsubstitution eliminates asparagine-glycine pairs, which form potentialdeamidation sites, by altering one or both of the residues. In anotherexample, the C-terminal lysine of the heavy chain of a PCSK9 antibody ofthe invention can be cleaved. In various embodiments of the invention,the heavy and light chains of the PCSK9 antibodies may optionallyinclude a signal sequence.

Once DNA fragments encoding the V_(H) and V_(L) segments of the presentinvention are obtained, these DNA fragments can be further manipulatedby standard recombinant DNA techniques, for example to convert thevariable region genes to full-length antibody chain genes, to Fabfragment genes, or to a scFv gene. In these manipulations, a V_(L)- orV_(H)-encoding DNA fragment is operatively linked to another DNAfragment encoding another protein, such as an antibody constant regionor a flexible linker. The term “operatively linked”, as used in thiscontext, is intended to mean that the two DNA fragments are joined suchthat the amino acid sequences encoded by the two DNA fragments remainin-frame.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH1,CH2 and CH3). The sequences of human heavy chain constant region genesare known in the art (see e.g., Kabat, E. A., et al., 1991, Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publ. No. 91-3242) and DNA fragmentsencompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG2 constant region. The IgG constant region sequence can beany of the various alleles or allotypes known to occur among differentindividuals, such as Gm(1), Gm(2), Gm(3), and Gm(17). These allotypesrepresent naturally occurring amino acid substitution in the IgG1constant regions. For a Fab fragment heavy chain gene, theV_(H)-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region. The CH1 heavy chainconstant region may be derived from any of the heavy chain genes.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, C_(L). The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabat,E. A., et al., 1991, Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIH Publ.No. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region. The kappa constant region maybe any of the various alleles known to occur among differentindividuals, such as Inv(1), Inv(2), and Inv(3). The lambda constantregion may be derived from any of the three lambda genes.

To create a scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (Seee.g., Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc.Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature348:552-554. The single chain antibody may be monovalent, if only asingle V_(H) and V_(L) are used, bivalent, if two V_(H) and V_(L) areused, or polyvalent, if more than two V_(H) and V_(L) are used.Bispecific or polyvalent antibodies may be generated that bindspecifically to PCSK9 and to another molecule.

In another embodiment, a fusion antibody or immunoadhesin may be madethat comprises all or a portion of a PCSK9 antibody of the inventionlinked to another polypeptide. In another embodiment, only the variabledomains of the PCSK9 antibody are linked to the polypeptide. In anotherembodiment, the V_(H) domain of a PCSK9 antibody is linked to a firstpolypeptide, while the V_(L) domain of a PCSK9 antibody is linked to asecond polypeptide that associates with the first polypeptide in amanner such that the V_(H) and V_(L) domains can interact with oneanother to form an antigen binding site. In another preferredembodiment, the V_(H) domain is separated from the V_(L) domain by alinker such that the V_(H) and V_(L) domains can interact with oneanother. The V_(H)-linker-V_(L) antibody is then linked to thepolypeptide of interest. In addition, fusion antibodies can be createdin which two (or more) single-chain antibodies are linked to oneanother. This is useful if one wants to create a divalent or polyvalentantibody on a single polypeptide chain, or if one wants to create abispecific antibody.

In other embodiments, other modified antibodies may be prepared usingPCSK9 antibody encoding nucleic acid molecules. For instance, “Kappabodies” (Ill et al., 1997, Protein Eng. 10:949-57), “Minibodies” (Martinet al., 1994, EMBO J. 13:5303-9), “Diabodies” (Holliger et al., 1993,Proc. Natl. Acad. Sci. USA 90:6444-6448), or “Janusins” (Traunecker etal., 1991, EMBO J. 10:3655-3659 and Traunecker et al., 1992, Int. J.Cancer (Suppl.) 7:51-52) may be prepared using standard molecularbiological techniques following the teachings of the specification.

Bispecific antibodies or antigen-binding fragments can be produced by avariety of methods including fusion of hybridomas or linking of Fab′fragments. See, e.g., Songsivilai & Lachmann, 1990, Clin. Exp. Immunol.79:315-321, Kostelny et al., 1992, J. Immunol. 148:1547-1553. Inaddition, bispecific antibodies may be formed as “diabodies” or“Janusins.” In some embodiments, the bispecific antibody binds to twodifferent epitopes of PCSK9. In some embodiments, the modifiedantibodies described above are prepared using one or more of thevariable domains or CDR regions from a human PCSK9 antibody providedherein.

Generation of Antigen-Specific Antibodies

More than 500 polyclonal and monoclonal antibodies raised againstrecombinant full-length human PCSK9, recombinant full length mousePCSK9, and various synthetic peptides were evaluated for their abilityto down regulate total LDLR protein in cultured Huh7 human liver cells.Among these antibodies were a set of antibodies raised to and reactivewith a set of 12-20 amino acid residue polypeptides that, based on thestructure of PCSK9, were predicted to cover majority of the proteinsurface. At the highest concentration, the best antibodies exhibitedonly about 60% blocking activity.

Thus, an alternative and heretofore unexplored approach was employed,namely, the generation of monoclonal antibodies by immunizing PCSK9 nullmice with recombinant full-length PCSK9 protein. This manner of antibodypreparation yielded antagonist antibodies that show complete blocking ofPCSK9 binding to LDLR, complete blocking of PCSK9-mediated lowering ofLDLR levels in Huh7 cells, and lowering of LDLc in vivo including inmice to levels comparable to that seen in PCSK9−/− mice, as shown inExample 7.

Representative antibodies (hybridomas) of the present invention weredeposited in the American Type Culture Collection (ATCC) on Feb. 28,2008, and were assigned the accession numbers in Table 3. Hybridomaswere deposited for antibodies 4A5, 5A10, 6F6 and 7D4.

TABLE 3 Antibody Reference ATCC Accession No. 4A5 PTA-8985 5A10 PTA-89866F6 PTA-8984 7D4 PTA-8983

EXAMPLES Example 1 Generating and Screening PCSK9 Antagonist AntibodiesGeneral Procedures for Immunization of Animals for Generating MonoclonalAntibodies:

Balb/c or 129/b16 pcsk9−/− mice were injected 5 times on a 13 dayschedule with 100 μg antigen. PCSK9−/− (that is, null or knock-out mice)can be obtained from, or as described by, Rashid et al., 2005, Proc NatlAcad Sci USA 102: 5374. See also U.S. Pat. No. 7,300,754. For the first4 injections, antigen was prepared by mixing the recombinant proteinswith adjuvant. Immunogen was given via injection to the scruff of theneck, the foot pads and intraperitoneally, approximately every 3 daysover the course of 11 days, with the last boost administered i.v.,without adjuvant. On Day 13, the mice were euthanized and their spleenswere removed. Lymphocytes were immortalized by fusion with anestablished cell line to make hybridoma clones using standard hybridomatechnology, distributed into 96 well plates. Clones were allowed togrow, then selected by ELISA screening using the immunizing antigen, asbelow.

ELISA Screening of Antibodies:

Supernatant media from growing hybridoma clones were screened separatelyfor their ability to bind the recombinant human PCSK9 or recombinantmouse PCSK9. The assays were performed with 96-well plates coatedovernight with 100 μl of a 1 μg/ml solution of one of the antigens.Excess reagents were washed from the wells between each step with PBScontaining 0.05% Tween-20. Plates were then blocked with PBS containing0.5% BSA. Supernatant was added to the plates and incubated at roomtemperature for 2 hours. Horse radish peroxidase (HRP) conjugatedgoat-anti mouse Fc was added to bind to the mouse antibodies bound tothe antigen. Tetramethyl benzidine was then added as substrate for HRPto detect the amount of mouse antibody present in the supernatant. Thereaction was stopped and the relative amount of antibody was quantifiedby reading the absorbance at 450 nm. Hybridoma clones that secretedantibodies that are capable of binding to either mouse or human PCSK9were selected for further analysis.

PCSK9-Mediated LDLR Down-Regulation in Huh7 Cells:

Hybridoma clones secreting human or mouse PCSK9 binding antibodies wereexpanded and supernatants were harvested. Total IgGs were purified fromapproximately 10 ml of the supernatant using protein A beads, dialyzedinto PBS buffer, and the final volume reduced to yield solutions with0.7-1 mg/ml of antibodies. Purified antibodies were then used to testtheir ability to inhibit the ability of PCSK9 to mediate LDLRdown-regulation in Huh7 cells. Huh7 cells were plated and allowed togrow to 80% confluency in RPMI media containing 10% FBS, 4 mM glutamine,and penicillin and streptavidin in 96 well plates. The medium waschanged to one containing 10% de-lipidated FBS for 8-16 hrs to induceLDLR expression. Cells were then incubated for 8-16 hours with 40μl/well of 293 expression media supplemented with 6 μg/ml of human(preferably) or mouse PCSK9, with or without 70-100 μg/ml of testantibodies. The PCSK9 and antibody containing media were removed at theend of incubation, and cells were lysed with 17 μl lysis buffer byshaking at 4 C for an hour. The lysis buffer consisted of 50 mM glycerolphosphate, 10 mM HEPES pH 7.4, 1% Triton X-100, 20 mM NaCl, and acocktail of protease inhibitors (Roche). Cell lysates were collected andanalyzed for LDLR protein levels via staining of Western blots followingSDS polyacrylamide gel electrophoresis. Hybridoma clones producingantibodies that can partially or fully rescue LDLR level were selectedfor further analysis. By “LDLR down regulation assay” is meant the aboveassay using Huh7 cells.

FIG. 1 illustrates the effect of anti-PCSK9 antagonistic monoclonalantibodies 7D4.4, 4A5.G3, 6F6.G10.3 and 5A10.B8 on the ability of humanand mouse PCSK9 to down regulate LDLR in cultured Huh7 cells. 100 nM ofmouse or human recombinant PCSK9, and a serial dilution of 25-800 nM ofantibodies were used. A) mouse PCSK9. B) human PCSK9. The figures areWestern blots showing that the antibodies are in general more effectivein blocking the function of human PCSk9 than mouse PCSK9. The severalantibodies have generally similar affinities for human PCSK9 but vary intheir affinity for murine PCSK9.

Example 2 Determining Antibody Binding Affinity

The affinities of PCSK9 antibodies to PCSK9 were measured on a surfaceplasmon resonance Biacore 3000 biosensor equipped with a research-gradesensor chip using HBS-EP running buffer (Biacore AB, Uppsala, Sweden—nowGE Healthcare). Rabbit polyclonal anti-Ms IgGs were amine-coupled atsaturating levels onto the chip using a standardN-hydroxysuccinimide/ethyldimethylaminopropyl carbodiimide (NHS/EDC)chemistry. The buffer was switched to HBS-EP+1 mg/mL BSA+1 mg/mLCM-dextran. Full-length PCSK9 IgGs were diluted to about 15 μg/mL andcaptured for 1 min at 5 μL/min to give levels of about 500RU per flowcell, leaving one blank to serve as a reference channel. 3.73-302 nMhPCSK9 or 2.54-206 nM mPCSK9 were injected as a 5-membered 3-fold seriesfor 1 min at 100 μL/min. Dissociation was monitored for 5 min. The chipwas regenerated after the last injection of each titration with two 30sec pulses of 100 mM phosphoric acid. Buffer cycles provided blanks fordouble-referencing the data, which were then fit globally to a simplebinding model using Biaevaluation software v.4.1. Affinities werededuced from the quotient of the kinetic rate constants(K_(D)=k_(off)/k_(on)). The results of Example 2 are shown in Table 4.These data show that the antibodies have excellent affinity for murinePCSK9 or human PCSK9, as indicated.

TABLE 4 Inhibition of K_(on) for K_(off) for K_(D) for LDLR-PCSK9 PCSK9PCSK9 PCSK9 mAb ligand binding (IC₅₀) (1/Ms) (1/S) (nM) 4A5 human 0.4 nM6.66 × 10⁴ 1.89 × 10⁻⁴ 2.8 5A10 human 0.4 nM 8.47 × 10⁴ 8.55 × 10⁻⁵ 16F6 human 1.5 nM 9.15 × 10⁴ 5.84 × 10⁻⁴ 6.4 7D4 human 1.5 nM 1.25 × 10⁵7.94 × 10⁻⁴ 6.4 4A5 mouse 3 nM 1.41 × 10⁵  7.2 × 10⁻⁴ 5.1 5A10 mouse 3nM 1.27 × 10⁵ 4.89 × 10⁻⁴ 3.9 6F6 mouse 10 nM 1.11 × 10⁵ 1.97 × 10⁻³17.7 7D4 mouse 1 nM 3.92 × 10⁴ 5.23 × 10⁻⁴ 1.3

Example 3 Analysis of the Effect of PCSK9 Antibodies on PCSK9-LDLRInteraction

PCSK9 has been shown to bind LDLR with an affinity of 180 nM underneutral pH (Cunningham et al., 2007, Nat Struct Mol Biol, 14(5):413-9).Recombinant mouse or human PCSK9 protein was biotinylated using thePierce reagents following the manufacture's instructions. ELISA plates(Corning Mixisorb) were coated with a solution of 1 μg/ml recombinantLDLR extracellular domain (R&D Systems) in each well at 4 C overnight,blocked with 2% BSA+PBS for 2 hrs at room temperature, and then washed 5times with washing buffer (1×PBS+0.05% Tween-20). Wells were incubatedwith 50 μl of indicated concentrations of biotinylated PCSK9 protein for1 hr RT. LDLR-PCSK9 binding can be stabilized by adding 50 μl of 4%FDH+4% sucrose+PBS solution and incubate for 5 min. Wells were washed 5times with washing buffer, incubated with 1:2000 dilution of HRPconjugated Strepavidin (Invitrogen) for 1 hr at RT, washed 5 times withwashing buffer. TMB substrate was added to the wells, the solution wasincubated 20 to 30 min at RT, and the reaction was terminated using 1 Mphosphoric acid. Signals were read at 450 nm.

FIG. 2 illustrates the dose-response of anti-PCSK9 antagonist monoclonalantibodies 6F6.G10.3, 7D4.4, 4A5.G3, 5A10.B8, negative control antibody42H7, and PBS on blocking the binding of recombinant biotinylated humanPCSK9 and mouse PCSK9 to immobilized recombinant LDLR extracellulardomain in vitro. Part A) shows human PCSK9 binding to human LDLRextracellular domain and that 7D4, 4A5, 5A10, and 6F6 are effective inblocking binding, whereas 42H7 and PBS are not. Part B) shows mousePCSK9 binding to human LDLR extracellular domain.

The interaction can also be evaluated in free solution at neutral pH.FIG. 3 illustrates the dose-response of anti-PCSK9 monoclonal antagonistantibodies 6F6.G10.3, 7D4.4, 4A5.G3 and 5A10.B8 on blocking binding ofrecombinant biotinylated human PCSK9 (30 nM) to Europium labeledrecombinant LDLR extracellular domain (10 nM) in solution at neutral pHin vitro. This assay measures binding in free solution at neutral pH.

Example 4 Epitope Mapping/Binding of Antibodies Using the CrystalStructure of the L1L3:PCSK9 Complex, Biacore, and Mutagenesis

a. Crystal structure of the L1L3:PCSK9 complex. The residues wereidentified by calculating the difference in accessible surface areabetween the L1L3:PCSK9 crystal structure and PCSK9 structure alone.PCSK9 residues that show buried surface area upon complex formation withL1L3 antibody were included as a part of the epitope. The solventaccessible surface of a protein was defined as the locus of the centreof a probe sphere (representing a solvent molecule of 1.4 Å radius) asit rolls over the Van der Waals surface of the protein. The solventaccessible surface area was calculated by generating surface points onan extended sphere about each atom (at a distance from the atom centreequal to the sum of the atom and probe radii), and eliminating thosethat lie within equivalent spheres associated with neighboring atoms asimplemented in program AREAIMOL (Briggs, P. J., 2000, CCP4 NewsletterNo. 38, CCLRC, Daresbury).

The result of the crystal structure analysis are shown in FIG. 23. FIG.23A shows the crystal structure of the PCSK9 (light gray surfacerepresentation) bound to the L1L3 antibody (black cartoonrepresentation). The epitope for L1L3 binding to PCSK9 involves residues153-155, 194, 197, 237-239, 367, 369, 374-379 and 381 of the PCSK9 aminoacid sequence (SEQ ID NO:53). By comparison, the epitope for the LDLREGF domain binding to PCSK9 involves residues 153-155, 194, 238, 367,369, 372, 374-375, and 377-381 (Kwon et al., 2008, PNAS 105: 1820-1825).

b. Group antibodies and epitopes based on competition in PCSK9 binding.Full-length IgGs were amine-coupled to a CM5 sensor chip (three per chipat about 7000RU final), using a standard EDC/NHS-mediated amine-couplingchemistry. One flow cell was left unmodified to provide a referencechannel. Human-PCSK9 (100 nM) was premixed with an array of IgGs (final500 nM) and these complexes were injected over the chip using 1 mininjections at 10 μL/min. Antibodies that bind to competing epitopes willblock the binding of PCSK9 to the antibody immobilized on the chip.Alternatively, a classical sandwich approach was used by first injectinghuman-PCSK9 at 50 nM for 1 min at 10 μL/min (to tether it via the IgG onthe chip) and then binding an array of IgGs (final 500 nM each) for 2mins each. The immobilized IgGs were regenerated with a mild acid(Pierce gentle elution buffer+1 M NaCl). Antibodies directed to knowndifferent epitopes were used as controls for positive sandwich formationin this assay.

c. Structure-guided mutagenesis to map antibody binding epitopes. Basedon the crystal structure of PCSK9 and the likely involvement of D374 inLDLR binding (Cunningham et al., 2007, Nat Struct Mol Biol, 14(5):413-419), nineteen PCSK9 surface-residue mutants (F379A, 1369A, R194A,D374Y, D238R, T377R, K222A, R199A, F216A, R218A, R237A, D192R, D367R,R165A, R167A, A443T, A53V, 1474V, H449A) near or far from the positionof D374 were chosen for mutation to map the antibody binding epitopes.

d. Mutant and antibody production. The 19 single point mutants weregenerated from the previously described wild-type DNA construct(Cunningham et al., 2007, supra) using standard DNA techniques. Themutant proteins were expressed using transient transfection in HEK293Tcells and secreted into the cell media. The mutant proteins werepurified with the high-throughput AKTA Xpress system (GE Healthcare) byNi²⁺ and size-exclusion chromatography steps, using conditions similarto those described earlier. Protein concentrations were determined usingthe LabChip instrument (Bio-Rad). The PCSK9-blocking murine antibodies4A5, 7D4, 5A10 and 6F6 were expressed with transient transfection inHEK293F cells and purified with a protein G column eluted with 0.1 MGlycine buffer at pH 2.8 and neutralized into 1.0 M Tris at pH 9.0.

e. The regions of PCSK9 that are contacted by monoclonal antibodies 5A10and 7D4 (preparation described later herein) were determined by proteintomography (Sidec AB, Stockholm, Sweden). The loops at positions186-200, 371-379, 176-181, 278-283, 449-453, 402-406, and 236-245 ofPCSK9 were proximal to amino acid residues of the antibody. Thesequences corresponding to the loops are shown in Table 5, and in apreferred embodiment, the antagonists of the invention bind to one ormore of these sequences in PSCK9.

TABLE 5 PCSK9 SEQ ID Loops  Sequence NO. 186-200 DTSIQSDHREIEGRV 1236-245 GRDAGVAKGA 2 371-379 ASSDCSTCF 3 176-181 GGSLVE 4 278-283 QPVGPL5 449-453 HGAGW 6 402-406 AEPEL 7

f. Biacore binding of the mutants to immobilized LDLR. Recombinant LDLRextracelluar domain protein was immobilized onto a Biacore SA chip. Eachmutant protein was injected to the Biacore-3000 M) in duplicates at 25mM to 0.012 mM at five concentrations (from 1° C., with a running bufferof 50 mM Tris pH 7.5, 2 mM CaCl₂, 200 mM NaCl, 0.02% P20 and 1 mg/mlBSA. All the results fit nicely to a 1:1 binding kinetics model. Asexpected, mutation at residues in direct contact with the EGF-A domain(F379A, R194A, 1369A, T377R, D238R) significantly weakens (by 10-100fold) LDLR binding. Moreover, three mutants not in contact with EGF-A(R199A, R218A, K222A) showed weaker binding (5-15 fold). This newfinding suggests that they are involved in binding other domains ofLDLR. Overall, these experiments validate the integrity and activity ofthe mutants for subsequent epitope mapping experiments.

g. Binding of the mutants to immobilized 4A5, 7D4, 5A10 and 6F6antibodies. Biotinylated anti-PCSK9 antibodies were immobilized on SAchips using standard methods. Mutant binding experiments were performedusing Biacore 3000 at 25° C. with a running buffer of 50 mM Tris-HCl pH7.5, 150 mM NaCl and 0.02% P20. Mutants were tested at 333 nM or 111 nMconcentrations in duplicates, with the ones giving weakened bindingcompared to the wild-type as the residues involved in mAb binding(listed below).

mAb Binding Residues in Descending Order of Mutant Effects 4A5 R237,F379, 369, R194, R199 & D238 5A10 R194, R237, 1369, D238, R199 6F6 R237,R194, F379, D238, 1369, T377, R199 7D4 R237, R194, F379, I369, R199Example 5 Cloning and Sequencing of Antibodies

One million hybridoma cells were homogenized using the QIAshredder spincolumns and total RNA was extracted according to RNAeasy Micro kit fromQIAGEN. cDNA was synthesized using SuperScript III RT kit fromInvitrogen. Variable regions from the PCSK9 antibodies were cloned usingthe mouse IgG-Primer Sets from Novagen, which consist of degenerateprimers for cloning mouse IgG heavy chain genes and the mouse kappa orlambda light chains. PCR cycling conditions were the followings: 1 cycleat 92 C for 2 min; two cycles at 94 C for 30 sec, 44 C for 30 sec and 72C for 2 min; two cycles at 94 C for 30 sec, 46 C for 30 sec and 72 C for2 min; two cycles at 94 C for 30 sec, 48 C for 30 sec and 72 C for 2min; two cycles at 94 C for 30 sec, 50 C for 30 sec and 72 C for 2 min;two cycles at 94 C for 30 sec, 52 C for 30 sec and 72 C for 2 min;followed by 35 cycles at 94 C for 30 sec, 54 C for 30 sec and 72 C for45 sec. The resulting PCR products were cloned into Topo-TA cloningvector from Invitrogen and sequenced. The cloned antibody sequences wereconfirmed by N-terminal sequencing of the first 10 amino acids of theoriginal antibodies produced from ascites.

Example 6 Generation of Antigens for Immunization

Recombinant human PCSK9 protein was produced as reported Cunningham etal., 2007, Nat Struct Mol Biol, 14(5):413-9. To produce recombinantmouse PCSK9 protein, the cDNA of mouse PCSK9 was cloned into mammalianexpression vector PRK5 with the addition of a 6-His tag at theC-terminus by methods known in the art, transiently transfected andexpressed in HEK293 cells. Recombinant protein was purified fromconditioned media using a Ni column.

Surface peptides of human and mouse PCSK9 were selected based on PCSK9protein structure, and synthesized by Elim Biopharmaceuticals.

Example 7 PCSK9-Specific Antibodies as PCSK9 Antagonists 1.Identification of PCSK9-Specific Antagonist Antibodies

a. Identification of PCSK9-Blocking Antibodies

Murine antibodies to human and/or mouse PCSK9 were generated byimmunizing mice with human-PCSK9 and mouse-PCSK9 synthetic peptides asprepared in Example 6 or recombinant proteins, and screening antibodiesby ELISA assay using human and/or mouse PCSK9 recombinant protein as theantigens as described in Example 1 and other standard hybridomaprocedures. Over 500 positive clones were obtained and allowed to growto confluency in 6 well plates with 10 ml media. Media supernatant werecollected and total IgGs in the conditioned media were purified usingmAb Select (Pierce). The ability of purified and concentrated mouse IgGsto inhibit mouse and human PCSK9 function was tested in Huh7 cells usingthe methods described in Example 1. Hybridoma clones expressing IgGsthat showed some degrees of blocking were expanded and retested. 60promising clones were subcloned, expanded, and injected into eitherBalb/c or nude mice to produce ascites. Antibodies purified from ascitesfluid were retested for their ability to inhibit the down regulation ofLDLR by human or mouse PCSK9 in Huh7 cells. Four hybridoma clones, 4A5,5A10, 6F6, and 7D4, were identified as being able to completely inhibithuman PCSK9 function, and at least partially inhibit mouse PCSK9function. To determine IC₅₀ of each of these blocking antibodies, aserial dilution of IgGs were used in the assay, starting from 100 μg/mlto 3.125 μg/ml, with human and mouse PCSK9 concentration being constantat 6 μg/ml.

b. Effect of PCSK9 Antagonists on PCSK9-LDLR Binding

PCSK9 has been shown to be co-localized with LDLR in cellularcompartments (Lagace et al., 2006, J Clin Inv, 116(11):2995-3005.Recombinant PCSK9 protein also binds to LDLR extracellular domain invitro (Fisher et al., 2007, JBC, 282(28):20502-12. To determine therelationship between inhibition of PCSK9 mediated down-regulation ofLDLR and inhibition of PCSK9-LDLR binding by antibodies, we tested thePCSK9 antibodies that partially or completely blocked PCSK9 function onLDLR and representatives of antibodies that do not block. All partialantagonistic antibodies also partially inhibited LDLR extracellulardomain binding to PCSK9, except one. Antagonistic antibodies that cancompletely block PCSK9 function, namely 4A5, 5A10, 6F6 and 7D4 alsocompletely inhibited LDLR extracellular domain binding to PCSK9 (Table5). IC₅₀ values of these four antibodies correlates with their bindingaffinity to PCSK9.

c. Epitope Determination of the Blocking Antibodies

FIG. 4 illustrates the epitope binning of anti-PCSK9 antibodies. Part A)shows epitope information of anti-PCSK9 mAbs, determined by binding tosynthetic 13-18-mer peptides or epitope binding via Biacore. Part B)shows the ability of immobilized antibodies 6F6, 5A10 and 4A5 to bind tohuman PCSK9 premixed with the mAbs indicated on the y axis by Biacoreassay.

Another monoclonal anti-PCSK9 antibody, termed 6G7, binds to recombinantmouse PCSK9 but not human PCSK9. See Table 6. 6G7, 4A5, 5A10, 6F6, and7D4 mutually exclude each other's binding to mouse PCSK9. Chimeraanalysis between mouse and human PCSK9 reveals that 6G7 binding to PCSK9requires the catalytic domain. See Table 6. Thus the binding sites of4A5, 5A10, 6F6, and 7D4 overlap the catalytic site and/or the epitopebound by 6G7.

TABLE 6 Recombinant protein 6G7 binding Human PCSK9 No Human pro + humancatalytic + mouse C-term No Human pro + mouse catalytic + mouse C-termYes Mouse pro + human catalytic + human C-term No Mouse Pro + mousecatalytic + human C-term Yes Mouse PCSK9 yesd. Determining Sequences Species Specificity of Anti-PCSK9 Antibodies

To determine the species specificity of the anti-PCSK9 antibodies,antibodies were incubated with plasma from different species and theresultant complexes were purified and probed by an independent antiPCSK9 antibody on Western blots. The antibodies 4A5, 5A10, 6F6, and 7D4recognized human, cynomolgus monkey, mouse, and rat PCSK9. See FIG. 5.Antibody 6G7 recognized only murine PCSK9 and an unrelated controlantibody 42H7 did not recognize any tested PCSK9. Id.

e. Determining Sequences of Antagonist PCSK9 Antibodies

The amino acid sequences of the variable domains of PCSK9 antibodies4A5, 5A10, 6F6, and 7D4 were determined using the method described inExample 5. The sequences indicate that the antibodies are related butdifferent from each other. Table 1 shows the amino acid sequences of thevariable regions of each antibody. Table 7 shows the CDR sequences ofthe light chains and heavy chains of Table 1 as identified by the Kabatand Chotia methods.

TABLE 7 Blocking PCSK9 Antibodies and Antigen-binding CDR Sequencesaccording to Kabat (underlined) and Chotia (bold). VL CDR1 VL CDR2VL CDR3 4A5 KASQNVGTNVA SASYRYS QQFYSYPYT (SEQ ID NO: 27)(SEQ ID NO: 28) (SEQ ID NO: 29) 5A10 KASQDVSTAVA SASYRYT QQRYSTPRT(SEQ ID NO: 30) (SEQ ID NO: 12) (SEQ ID NO: 31) 6F6 SASQGISNYLN YTSSLHSQQYSKLPFT (SEQ ID NO: 32) (SEQ ID NO: 33) (SEQ ID NO: 55) 7D4KASQDVSNALA SASYRYT QQHYSTPWT (SEQ ID NO: 34) (SEQ ID NO: 12)(SEQ ID NO: 35) L1L3 RASQGISSALA SASYRYT QQRYSLWRT (SEQ ID NO: 11)(SEQ ID NO: 12) (SEQ ID NO: 13) VH CDR1 VH CDR2 VH CDR3 4A5 GYTFT DYYMNDINPNNGGTTYNQKFKG WLLFAY (SEQ ID NOs: (SEQ ID NOs: 38 and 39)(SEQ ID NO: 40) 56(whole), 36 and 37) 5A10 GYTFT SYWMH EINPSNGRTNYNEKFKSERPLYAMDY (SEQ ID NOs: (SEQ ID NO: 43 and 44) (SEQ ID NO: 45)57(whole), 41 and 42) 6F6 GYTFT DYYMN DINPNNGGTSYNQKFKG GGIYYRYDRNYFDY(SEQ ID NOs: (SEQ ID NO: 38 and 46) (SEQ ID NO: 47)56(whole), 36 and 37) 7D4 GFTFS DYYMA NINYDGSNTSYLDSLKS EKFAAMDY(SEQ ID NOs: (SEQ ID NOs: 50 and 51) (SEQ ID NO: 52)58(whole), 48 and 49) L1L3 GYTFT SYYMH EISPFGGRTNYNEKFKS ERPLYASDLSEQ ID NOs: (SEQ ID NO: 9 and 61) (SEQ ID NO: 10) 59(whole), 60, and 8.Anti-PCSK9 IgGs 4A5, 5A10 and 6F6 were amine coupled to the Biacorechip. hPCSK9 (100 nM) was mixed with 500 nM of 4A5, 5A10, 6F6 or 7D4 invarious ratios and injected for 1 min at 10 μl/min. The four antibodiesmutually blocked one another irrespective of the assay orientationtested, suggesting that they all bind to competing epitopes. Incontrast, they are able to form sandwich complexes with othernon-fully-blocking antibodies that were mapped to specific regions usingsynthetic peptides.

2. Effect of PCSK9 Specific Antibodies as PCSK9 Antagonist In Vivo

a. PCSK9 Antagonist Antibodies Lower Serum Cholesterol in Mice

To determine if PCSK9 antagonist monoclonal antibodies can affectcholesterol levels in vivo by inhibiting the function of extracellularPCSK9, the effect of 7D4 was tested against mouse PCSK9 in vitro, onserum cholesterol when injected into mice. 6 to 7 week old male 057/b16mice were kept on a 12 hr light/dark cycle, bled to collectapproximately 70 μl serum on day −7. Antagonist PCSK9 antibody 7D4, anda control isotype matching monoclonal antibody were injected into male 7week old C57/b16 mice via i.p. injections on days 0, 1, 2, and 3. Micewere sacrificed on day 4 without fasting, and serum samples werecollected. All frozen serum samples were sent to IDEXX laboratories fortotal cholesterol, triglyceride, HDL cholesterol and LDL cholesterolmeasurements. FIG. 6 shows that 7D4 lowered serum cholesterol by 48%,while the control antibody did not have any significant affect. Both theamount and percentage of reduction are similar to what was reported forPCSK9−/− mice (PCSK9 knock-out mice), suggesting that one can achievecomplete or near complete inhibition of PCSK9 function through blockingextracellular PCSK9 only, and that intracellular PCSK9 plays little orno role in down-regulating LDLR under normal physiological conditions.As expected, liver LDLR levels were induced in animals treated with 7D4compared to those treated with a control antibody (FIG. 6).

b. A Partially Blocking Antibody Had No Effect on Blood CholesterolLevels

FIG. 7 illustrates that a partial antagonist polyclonal anti-PCSK9 mAbCRN6 does not affect cholesterol levels in mice. Two groups of 8 weekold C57/b16 mice (n=10 mice/group) were bled and tested for cholesterollevels on day -7; dosed with 15 mg/kg/day of CRN6 or a control antibodyby i.v. administration on days 0, 1, 2 and 3; and then bled and testedfor cholesterol levels 24 hrs after the final dose. FIG. 7A shows thatCRN6 antibody partially blocks PCSK9 mediated down regulation of LDLR inHuh7 cells in vitro. FIG. 7B shows that administration of CRN6 antibodydoes not affect serum cholesterol levels in mice.

c. Prolonged Effect on Serum Cholesterol by Antagonist PCSK9 mAb inMice.

A time course study was performed to determine the time of onset andduration of the cholesterol lowering effect of PCSK9 antagonistantibodies in mice. MAb 7D4 or saline control were each injected i.v. at10 mg/kg or 3 ml/kg in 48 6-week-old C57/b16 mice. Eight mice from eachtreatment group were sacrificed on days 1, 2, 4, 7, 14 and 21 afterinjection. A single injection of 7D4 produced a fast and prolongedlowering effect on serum cholesterol. A 25% reduction in serumcholesterol was seen at 24 hrs after injection. See FIG. 8. Maximum dropof serum cholesterol was observed at the 7 day time point. At 21 days,the reduction in cholesterol is no longer statistically significant.Part B) shows HDL cholesterol. LDL cholesterol levels were very low.

FIG. 9 illustrates that the anti-PCSK9 antagonist mAb 7D4 dosedependently reduces serum total cholesterol, HDL, and LDL in mice. Sixgroups of 8 week old 057/b16 mice (n=8/group) were bled and tested forbasal cholesterol levels on day -7 and administered with the indicateddoses of antibodies or saline on days 0, 1, 2, and 3 by i. p. bolusinjection. Serum samples were collected and tested for cholesterollevels 24 hrs after the last dose. FIG. 9A shows total cholesterollevels, which decreased to less than 60% of control after administrationof 3 to 30 mg/kg/day. The maximal effect on total cholesterol was seenat 10 mg/kg, and statistically significant reduction at 1 mg/kg. FIG. 9Bshows HDL levels, which decreased to less than 70% after administrationof 3 to 30 mg/kg/day. FIG. 9C shows LDL levels, which decreased tonearly zero at all tested doses of 0.3 mg/kg/day and above.

d. Dose Response of Antagonist Antibodies Specific to PCSK9 in Mice

FIG. 10 illustrates that anti-PCSK9 antagonist antibody 5A10 dosedependently lowers cholesterol levels in mice. FIG. 10A shows six groupsof 8 week old 057/b16 mice (n=8/group) to which were administered theindicated doses of antibodies or saline daily on days 0, 1, 2, and 3 byi.v. bolus injection. Serum samples were collected and tested forcholesterol levels 24 hrs after the last dose and showed a graduateddecrease with increasing dose of antibody. FIG. 10B shows five groups of8 week old C57/b16 mice (n=8/group) to which were administered theindicated doses of antibodies or saline on day 0 by i. p. bolusinjection. Serum samples were collected and tested for cholesterollevels on day 7 and also showed .a graduated decrease with increasingdoses of antibody.

FIG. 11 illustrates that anti-PCSK9 antagonist antibodies 4A5 and 6F6lower cholesterol levels in mice in a dose-dependent fashion. Eight weekold C57/b16 mice (n=8/group) were administered the indicated doses ofantibodies or saline on day 0 by i.p. bolus injection. Serum sampleswere collected and tested for cholesterol levels on day 7. In FIG. 11A,the antibody 4A5 showed a graduated decrease in total serum cholesterolwith increasing dose of antibody. In FIG. 11B, the antibody 6F6 showeddecrease in total serum cholesterol at 10 mg/kg/day.

Anti-PCSK9 antagonist antibodies 4A5, 5A10, 6F6 and 7D4 increase liverLDLR levels in mice as found by Western blot analysis. See FIG. 12. For4A5, 5A10 and 6F6, 8 week old C57/b16 mice were administered with 10mg/kg of antibodies or saline on day 0 by i.v. bolus injection, animalswere sacrificed on day 7, and whole liver lysate of 3 individual animalswere analyzed for LDLR and GAPDH protein levels by Western. For 7D4, 8week old B16/c57 mice were administered with 10 mg/kg of antibodies ondays 0, 1, 2, and 3 via i. p. bolus injection, animals were sacrificedon day 4, and whole liver lysate of 3 individual animals were analyzedfor LDLR and GAPDH protein levels by Western blot. All antibody-treatedmice showed high levels of LDLR as compared to the PBS control mice.

FIG. 13 illustrates that anti-PCSK9 antagonist antibody has no effect inthe LDLR−/− mouse. Eight week old LDLR−/− mice (LDLR KO mice) wereadministered 10 mg/kg 4A5 or saline on day 0 by i.p. bolus injection.Serum samples (from n=9-10 mice) were collected and tested forcholesterol levels on day 7. Administration of the antibody did notappreciably alter the levels of total serum cholesterol, HDL, or LDL.

FIG. 14 illustrates that multiple treatments of anti-PCSK9 antagonistantibodies in mice can substantially decrease total serum cholesterol.Eight week old C57/b16 mice were administered the indicated doses ofantibodies or PBS on days 0, 7, 14 and 21 by i.v. bolus injection. Serumsamples (n=5-11 mice) were collected and tested for cholesterol levelson day 28.

Example 8 PCSK9 Antagonist Antibodies Lower Serum LDL in Non-HumanPrimates

To test the in vivo effect of antibodies to PCSK9, antibody 7D4 wastested in cynomolgus monkeys. Four 3-4 year old cynomolgus monkey wereinjected with vehicle (PBS+0.01% Tween 20) on day 0, and 10 mg/kg 7D4 onday 7. Plasma lipid profiles were analyzed on days 0, 2, 7, 9, 11, 14,21 and 28 following overnight fasting. A single injection of 10 mg/kg7D4 produced a dramatic reduction in plasma LDL (60%) (FIG. 15A) and LDLparticle numbers (FIG. 15D) in all 4 animals, while having minimaleffect on their HDL levels (FIG. 15B) and HDL particle numbers (FIG.15E). Total cholesterol (FIG. 15C) was also reduced following 7D4treatment, while triglyceride level (FIG. 15F) was not significantlyaffected. Total 7D4 (G), and total PCSK9 levels (H) were also measured.

FIGS. 16A-D illustrate the dose-response of anti-PCSK9 antibody 7D4 onserum cholesterol levels in the cynomolgus monkey. Two male and twofemale cynomolgus monkeys 3-5 years of age in each group were given theindicated dose of 7D4 on day 7 and an equal volume of saline on day 0 byi.v. bolus injection. Plasma samples were taken at indicated time pointsand plasma LDL levels were measured.

FIG. 17 illustrates a comparison of anti-PCSK9 antibodies 4A5 (FIG.17A), 5A10 (FIG. 17B), 6F6 (FIG. 17C) and 7D4 (FIG. 17D) on serumcholesterol levels in the cynomolgus monkey. Two male and two femalecynomolgus monkeys 3-6 years of age in each group were given 1 mg/kg ofthe indicated antibody on day 0 by i.v. bolus injection. Plasma sampleswere taken at indicated time points, plasma LDL levels were measured andnormalized to that on day -2.

FIG. 18 illustrates the effect of anti-PCSK9 antagonist antibody 7D4 onplasma cholesterol levels of cynomolgus monkeys fed a 33.4% kcal fatdiet supplemented with 0.1% cholesterol. Six 3-5 year old cynomolgusmonkeys were put on high-fat diet for 16 weeks. Three monkeys weretreated with 10 mg/kg 7D4 and three with saline on the indicated date.LDL levels of individual monkeys were measured and normalized to that ofthe treatment day.

Example 9 Humanized Anti-PCSK9 Antibody

The murine monoclonal antibody 5A10 was humanized and affinity maturedto provide the L1L3 antibody. L1L3 has an affinity for murine PCSK9 of200 pM and an affinity for human PCSK9 of 100 pM when measured byBiacore. L1L3 completely inhibits the PCSK9-mediated down regulation ofLDLR in cultured Huh7 cells when incubated with 100 nM human or murinePCSK9 antibody. See FIG. 19.

FIG. 20 illustrates the dose-response of L1L3, mouse precursor 5A10, andnegative control antibody 42H7 to block the binding of recombinantbiotinylated human PCSK9 and mouse PCSK9 to immobilized recombinant LDLRextracellular domain in vitro. FIG. 20A shows human PCSK9 binding tohuman LDLR extracellular domain at pH 7.5. FIG. 20B shows human PCSK9binding to human LDLR extracellular domain at pH 5.3. FIG. 20C showsmouse PCSK9 binding to human LDLR extracellular domain at pH 7.5. FIG.20D shows mouse PCSK9 binding to human LDLR extracellular domain at pH5.3.

FIG. 21 shows the effect on serum cholesterol of treatment with 10 mg/kgL1L3 in mice. Two groups (n=8/group) of 8 week old 057/b16 mice weredosed with 10 mg/kg L1L3 or an equal volume of saline by i. p. injectionon day 0. Serum samples were collected and assayed for cholesterollevels on days 2, 4 and 7. L1L3 decreased total serum cholesterol byabout 40% at days 2 and 4. In another study, when 10 mg/kg of L1L3 wasadministered as a single intraperitoneal (IP) dose to C57BL/6 mice fed anormal diet (n=10), serum cholesterol levels were reduced by 47%compared to saline treated controls, 4 days post treatment. When L1L3was administered as a single IP dose at 0, 0.1, 1, 10 and 80 mg/kg(n=6/group) in a dose-response experiment in male Sprague-Dawley ratsfed a normal diet, serum cholesterol levels were dose-dependentlyreduced, with maximum effect of 50% seen at 10 and 80 mg/kg, 48 hourspost dosing. The duration of the cholesterol repression was also dosedependent, ranging from 1 to 21 days.

The amino acid sequence of L1L3 fully humanized heavy chain (SEQ IDNO:15) is shown in Table 8. The sequence of the variable region isunderlined (SEQ ID NO: 54).

TABLE 8 qvqlvqsgae vkkpgasvkv sckasqytft syymhwvrqapgqglewmge ispfqgrtny 60 nekfksrvtm trdtststvy melsslrsed tavyycarerplyasdlwgq gttvtvssas 120 tkgpsvfpla pcsrstsest aalgclvkdy fpepvtvswnsgaltsgvht fpavlqssgl 180 yslssvvtvp ssnfgtqtyt cnvdhkpsnt kvdktverkccvecppcpap pvagpsvflf 240 ppkpkdtlmi srtpevtcvv vdvshedpev qfnwyvdgvevhnaktkpre eqfnstfrvv 300 svltvvhqdw lngkeykckv snkglpssie ktisktkgqprepqvytlpp sreemtknqv 360 sltclvkgfy psdiavewes ngqpennykt tppmldsdgsfflyskltvd ksrwqqgnvf 420 scsvmhealh nhytqkslsl spgk 444

The amino acid sequence of L1L3 fully humanized light chain (SEQ IDNO:14) is shown in Table 9. The variable region is underlined (SEQ IDNO: 53).

TABLE 9 diqmtqspss lsasvgdrvt itcrasqgis salawyqqkpgkapklliys asyrytgvps 60 rfsgsgsgtd ftftisslqp ediatyycqq ryslwrtfgqgtkleikrtv aapsvfifpp 120 sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnSqesvteqdskd styslsstlt 180 lskadyekhk vyacevthqg lsspvtksfn rgec 214

FIG. 22 shows the effect of intravenous administration of an effectivedose (3 mg/kg) of antibody 5A10 (solid circles) or antibody L1L3 (solidsquares) to each of four cynomolgus monkeys at day zero. The change inserum HDL (FIG. 22A) and serum LDL (FIG. 22B) was measured from −2 to+28 days. Both antibodies resulted in greater than about 70% decrease inserum LDL levels by about seven days, an effect that substantiallypersisted for about six more days in the animals administered L1L3. Allthe animals showed normal liver and kidney function and near-normalhematocrits.

L1L3 dose-dependently reduced LDL-C, with a maximum effect observed inthe 10 mg/kg group, which maintained a 70% reduction in LDL-C levelsuntil day 21 post-dosing, and fully recovered by day 31. HDL-C levelswere not affected by L1L3 treatment in all dose groups. The animals inthe 3 mg/kg dose group (n=4) were also given two additional IV doses of3 mg/kg L1L3 on study days 42 and 56 (2-weeks apart). These twoadditional doses again lowered LDL-C and maintained LDL-C levels below50% for 4 weeks. LDL-C levels returned to normal two weeks later. SerumHDL-C levels remained unchanged throughout the study.

The efficacy of L1L3 in non-human primates with hypercholesterolemia andpharmacodynamic interactions between L1L3 and HMG-CoA reductaseinhibiting statins were investigated. Prior to the initiation of thestudy, the LDL-C levels of a cohort of cynomolgus monkeys (n=12) wereelevated to an average of 120 mg/dL, compared to the normal averagelevels of 50 mg/dL, by feeding with a diet containing 35% fat (wt/wt)and 600 ppm cholesterol for over 18 months. Surprisingly, no effect wasobserved on serum total cholesterol or LDL-C levels after the dailyadministration of a medium-dose (10 mg/animal) of Crestor® (rosuvastatincalcium) for 6 weeks, and after a subsequent daily administration ofhigh-dose (20 mg/kg) for 2 weeks. A single administration of 3 mg/kgL1L3 with Crestor® or vehicle treatment for 2 weeks, effectively loweredserum LDL-C levels by 56% by day 5 post treatment, and graduallyrecovered in 2.5 to 3 weeks while not affecting HDL-C levels. Uponswitching the animals to daily administration of 50 mg/kg Zocor®(simvastatin), their LDL-C levels reached a maximal reduction of 43% atday 5, and stabilized thereafter. After 3 weeks of 50 mg/kg/day Zocor®administration, these animals were treated with a single dose of 3 mg/kgL1L3 while still receiving 50 mg/kg/day Zocor®. Administration of L1L3resulted in another additional 65% reduction in LDL-C, in addition tothe 43% reduction by Zocor®, by day 5, and returned to pre-dosing levelswithin 2 weeks.

Other CDR amino acid substitutions were made to 5A10 in the course ofhumanization and affinity maturation and to achieve particularproperties. The sequences of the modified CDRs and the PCSK9 bindingabilities of the antibodies containing these modified CDRs are listed inFIGS. 24 A-G. The numbers following each sequence in FIGS. 24 A-Grepresent the SEQ ID NO for that sequence.

The disclosures of all references cited herein are hereby incorporatedby reference herein.

It is claimed:
 1. An antagonist of PCSK9 which comprises an isolatedantibody, a peptide, or an aptamer which interacts with PCSK9 and, whenadministered to a subject, lowers the blood low density lipoprotein(LDL) cholesterol in said subject.
 2. The antibody of claim 1, whereinthe antibody specifically binds to PCSK9 and which is a full antagonistof the PCSK9-mediated effect LDL receptor (LDLR) levels as measured invitro using an LDLR down-regulation assay in Huh7 cells.
 3. The antibodyof claim 2, wherein the antibody is humanized, human, or chimeric. 4.The antibody of claim 2, wherein the antibody recognizes an epitope onhuman PCSK9 comprising amino acid residues 153-155, 194, 195, 197,237-239, 367, 369, 374-379 and 381 of the PCSK9 amino acid sequence ofUniprot Accession Number Q8NBP7.
 5. The antibody of claim 5, wherein theantibody epitope on human PCSK9 does not comprise one or more of aminoacid residues 71, 72, 150-152, 187-192, 198-202, 212, 214-217, 220-226,243, 255-258, 317, 318, 347-351, 372, 373, 380, 382, and 383 of thePCSK9 amino acid sequence of Uniprot Accession Number Q8NBP7.
 6. Theantibody of claim 2, wherein the antibody recognizes an epitope on humanPCSK9 that overlaps with more than about 75% of the surface on PCSK9that interacts with the EGF-like domain of the LDLR.
 7. An isolatedantibody that comprises a PCSK9 binding region that competes with, orrecognizes a first epitope of PCSK9 that overlaps with a second epitopethat is recognized by, a monoclonal antibody selected from the groupconsisting of 5A10, which is produced by a hybridoma cell line depositedwith the American Type Culture Collection and assigned accession numberPTA-8986; 4A5, which is produced by a hybridoma cell line deposited withthe American Type Culture Collection and assigned accession numberPTA-8985; 6F6, which is produced by a hybridoma cell line deposited withthe American Type Culture Collection and assigned accession numberPTA-8984, and 7D4, which is produced by a hybridoma cell line depositedwith the American Type Culture Collection and assigned accession numberPTA-8983.
 8. An isolated antibody comprising a light chain variableregion (VL) CDR1 having the amino acid sequence shown in SEQ ID NO:11, aVL CDR2 having the amino acid sequence shown in SEQ ID NO:12, and/or VLCDR3 having the amino acid sequence shown in SEQ ID NO:13, or a variantthereof having one or more conservative amino acid substitutions inCDR1, CDR2, and/or CDR3.
 9. The antibody of claim 8 further comprising aVH CDR1 having the amino acid sequence shown in SEQ ID NO:59 or 8, a VHCDR2 having the amino acid sequence shown in SEQ ID NO:61 or 9, and/orVH CDR3 having the amino acid sequence shown in SEQ ID NO:10, or avariant thereof having one or more conservative amino acid substitutionsin CDR1, CDR2, and/or CDR3.
 10. The antibody of claim 9, wherein thevariant of the VH CDR1 comprises a substitution at amino acid position 8of SEQ ID NO:59, a variant of the VH CDR2 comprises a substitution atone or more of amino acid positions 3, 4, 5, 6, and 7 of SEQ ID NO:9,and/or a variant of the VH CDR3 comprises a substitution at one or bothof amino acid positions 7 and 9 of SEQ ID NO:
 10. 11. A pharmaceuticalcomposition comprising a therapeutically effective amount of theantibody of claim
 2. 12. The pharmaceutical composition of claim 11,further comprising a therapeutically effective amount of a statin.
 13. Amethod for reducing a level of LDL-cholesterol in blood of a subject inneed thereof, comprising administering to the subject a therapeuticallyeffective amount of the antibody of claim 2.